The High Priest Oroe invites all to enter the temple, and Babylonians (along with others from abroad, including Idreno the Indian King) do so carrying offerings to Baal. Assur states that the day has come for the Queen to choose a successor and he reminds all of his own valour. Idreno express surprise at Assur's aspirations and all express their individual concerns and fears. Semiramide enters to the acclaim of all, but Idreno. Idreno, and Assur individually speculate as to who will be chosen. They press the queen to announce her decision, but at the same time Semiramide herself is fearful about making that decision, especially as she appears to be expecting someone's arrival. Suddenly, the temple is plunged into darkness and there is general consternation amidst fears of its imminent collapse. All desert the temple. Arsace, a warrior, enters. He has been told by his dying father to go to the temple in Babylon, and he was also urgently sent for by Semiramide. He brings with him a casket belonging to his father, but he is puzzled as to why he has been called back to Babylon. He declares his love for Princess Azema who loves him though she has been promised to the dead King Nino's lost son, Ninia. Arsace states his unwillingness to support Assur in his bid for the throne. Arsace asks to see the High Priest. Oroe enters, opens the casket, and exclaims upon seeing it that it contains the holy relics of the dead king. He hints to Arsace about some treachery had been involved. Seeing Assur approach, Oroe leaves with the relics. Assur arrives and questions the reason for Arsace's return. The two men discuss Azema, with Arsace reaffirming his love for her, while Assur states that he too loves her. "You have no idea what love is", the younger man tells the older.
Azema is happy that Arsace is now in Babylon. Idreno follows her and asks for her hand; she tells him that this must be Semiramide's decision. He assums that his rival can only be Assur. Scornfully told that it will never be Assur, Idreno is comforted, although he expresses his desire to punish the wicked boldness of a rival and continues to express desire for Azema.
Having fallen in love with Arsace and believing that he loves her, Semiramide waits for his arrival. She receives a message from the Oracle, telling her that a wedding will make a new king. She believes this to be a sign from the gods that they approve of her plans, and orders preparations for a wedding. When Arsace arrives, he alludes to his love for Azema without specifically naming her, but he also declares that he will die for his queen if necessary. Semiramide still believes that he really loves her, and vows that she will give him all he desires. They leave separately.
All enter to the Trone Room, awaiting Semiramide's arrival and her announcement of her choice of successor. Arsace, Idreno, Oroe, and Assur all swear to obey her command, no matter what she decides. She demands loyalty to the man she chooses and they are told that he will also be the queen's husband. When Semiramide names Arsace as her chosen one, Assur is outraged and Idreno accepts the decision but requests Azema's hand, which is granted. After asking Oroe to unite Semiramide and Arsace, Semiramide is horrified by the uproar which emits from the close-by tomb of King Nino. All are horrified as King Nino's ghost appears, warning of the crimes to be expiated, telling Arsace that he will reign and to respect the High Priest's wisdom, and commanding him to come down into his tomb. Each character expresses his or her own anguish.
In a brief encounter, Mitrane warns the royal guard to keep Assur under surveillance and not to allow him to leave the palace. Then Semiramide enters, followed shortly after by Assur. Conflict between the two soon emerges. She reminds him of that it was he who gave the cup of poison to Nino, thus causing his death, and he reminds her that it was she who had prepared it. Recalling that at that time she had a son, Ninia, she speculates that he might have been killed by the same man who killed Nino. Assur continues to pressure Semiramide to make him king. In turn, she threatens to reveal the crime. They recall the terror and retribution that each could inflict upon the other if the true facts came to light. Semiramide continues to demand that Assur acknowledge Arsace as his king. Rejoicing is heard in the distance, and while Semiramide regains some of her former happiness, Assur becomes resigned to his fate.
In King Nino's tomb, Oroe and the Magi assemble. The High Priest urges Arsace to come forward but makes him aware that there may be some unpleasant news awaiting him. Upon his arrival, Oroe tells him that he is Ninia, Nino's son, who had been saved by Fradate (one of the priests) and brought up as his own. Aghast at this news, Arsace then learns that Semiramide is his mother. To reinforce this news, Oroe hands him a scroll, writing by the King before his death, the reading of which confirms the Priest's statements. The final blow comes when Arsace reads Nino's words, and realises that his mother and Assur were the ones who killed his father. Almost collapsing in grief into Oroe's arms, he asks for comfort, but the priests quickly reinforce his need to take immediate revenge. They equip him with armour and a sword and give him the determination to proceed. Sword in hand, Arsace leaves.
Azema , alone with Mitrane in Semiramide's apartments, complains that she has lost everything now that Arsace, the love of her life, is due to marry the queen. Entering, Idreno overhears this and is distraught. Azema promises him her hand if he so desires it, but he wishes that she would love him. Maids, Lords, and Indians lead them all to the temple.
In the temple Semiramide confronts Arsace, who finally hands her the scroll which has revealed all. Horrified, she then understands Arsace's real identity, and becomes remorseful, offering herself to his revengeful blows. He swears filial loyalty, expressing the wish to spare his mother. Together, they each accept the reality, but Arsace declares that he must go to his father's tomb and take whatever action is necessary. Knowing what is in store, Semiramide urges him to "Return to me victorious".
Defiantly, Assur proclaims that this will be Arsace's last day on earth. Learning from his men that the people have turned against him, he still vows to kill Arsace. He moves towards the tomb only to find some unknown force, some apparition holding him back. His men urge him on, but still the apparition remains in his mind. His men are puzzled, until he seems to recover and then, with his men beside him, vows to fight on.. Along with Oroe, Arsace enters the tomb. He awaits his rival. Assur enters, also awaiting Arsace. Semiramide then comes in to pray at Nino's tomb, asking for forgiveness and protection for her son. In the confusion of the darkness, all three - Arsace, Semiramide, and Assur - express some bewilderment as to the loss of their courage at this crucial moment. But in the darkness and seeking to strike Assur, Arsace strikes Semiramide as she steps between them to stop the fight. Surprised to learn Arsace's real identity, Assur is arrested, Semiramide dies, and to general acclaim by the people, Arsace reluctantly accepts that he shall be King.