The island has been linked to the rest of Rome by two bridges since antiquity, and was once called Insula Inter-Duos-Pontes which means "the island between the two bridges". The Ponte Fabricio, the only original bridge in Rome, connects the island from the northeast to the Field of Mars in the rione Sant'Angelo (left bank). The Ponte Cestio, of which only some original parts survived, connects the island to Trastevere on the south (right bank).

There is a legend which says that after the fall of the hated tyrant Tarquinius Superbus (510 BC), the angry Romans threw his body into the Tiber. His body then settled onto the bottom where dirt and silt accumulated around it and eventually formed Tiber Island. Another version of the legend says that the people gathered up the wheat and grain of their despised ruler and threw it into the Tiber, where it eventually became the foundation of the island.

In ancient times, before Christianity spread through Rome, Tiber Island was avoided because of the negative stories associated with it. Only the worst criminals and the contagiously ill were condemned there. This however changed when a temple was built on the island.

Tiber Island was once the location of an ancient temple to Aesculapius, the Greek god of medicine and healing.

Accounts say that in 293 BC, there was a great plague in Rome. Upon consulting the Sibyl, the Roman Senate was instructed to build a temple to Aesculapius, the Greek god of healing, and sent a delegation to Epidauros to obtain a statue of the deity. The delegation went on board a ship to sail out and obtain a statue. Following their magical belief system, they obtained a snake from a temple and put in on board their ship. It immediately curled itself around the ship's mast and this was deemed as a good sign by them. Upon their return up the Tiber river, the snake slithered off the ship and swam onto the island. They believed that this was a sign from Aesculapius, a sign which meant that he wanted his temple to be built on that island.

This location may have been chosen for the Aesculapius Temple because it was separate from the rest of the city, which could help protect whoever was there from plague and illnesses.

The island eventually became so identified with the temple it supported that it was modeled to resemble a ship as a reminder of how it came to be. Travertine facing was added in mid or late first century by the banks to resemble a ship's prow and stern, and an obelisk was erected in the middle, symbolizing the vessel's mast. Walls were put around the island, and it came to resemble a Roman ship. Faint vestiges of Aesculapius' caduceus with an entwining snake is still visible on the "prow".

During summer, the island hosts the Isola del Cinema film festival.

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