The earliest traces of human inhabitants in the area date back to the late prehistoric period (3rd - 2nd Millenium B.C.), including a small settlement from the Villanovian Age (9th Century B.C.) at the base of the hill now known as San Pietro.
Tuscania was an important inland Etruscan town in the 6th Century B.C., benefitting from trade through the port of Villa Regis (Montalto di Castro). It became more independent the following century, basing its power on dominance of the local trade between the inland and the coast. In the 3rd Century B.C., the Romans built the Via Clodia, from Rome to Siena, which passed through Tuscania and led to the development of the city and the building of aqueducts, spas and housing. Tuscania officially became a Roman municipium in 88 B.C. and flourished for several centuries thanks to trade with the capital. In the 5th Century A.D. it became one of the first bishoprics of the Christian Church.

However, with the fall of the Roman Empire, this period of peace ended abruptly. In 569 the town was conquered by the Longobards and again in 774 by Charlemagne, who donated it to the Church. This was the origin of the Estate of San Pietro in Tuscia, one of the earliest belongings of the Papal State. The town became known as Toscanella (until 1911), apparently due to the inhabitants' rebellious nature (the name has undertones of minor importance, being a diminuitive). In fact, it was only under Pope Innocent III (1198-1216) that the local nobles finally recognised the authority of the Church.

In the 12th Century, it became a city state and the imposing city walls were built. After being defeated by Frederick II of Svevia, Tuscania experienced a period of peace and propserity, with the population rising to some 5,000. The town was then usurped by the Vico family in the 14th Century before it returned to the Church a century later thanks to Cardinal Giovanni Vitelleschi of Viterbo. As a result of the decision to concentrate all administrative matters in Viterbo and to prefer the Via Cassia (which runs by Viterbo) rather than the Via Clodia, Tuscania lost a lot of its economic and political clout over the following centuries. In 1495 Charles VIII sacked the city as he passed through.

The fortunes of Tuscania revived under Napoleon, when it was used as an administrative centre, though it never gained more than provincial standing. After Napoleon's defeat, Tuscania returned to the Church (firstly in the Diocese of Civitavecchia, then that of Viterbo in 831). The Campanari family made an important contribution to archaeological research during this period.
In 1870 Bixio took the town and Tuscania became part of the Province of Rome. In 1927 it became part of the Province of Viterbo.

In 1971 the city was devastated by a violent earthquake. Much of the town we see today has been rebuilt since then, preserving the original look, including the walls and its two dominating churches: San Pietro and Santa Maria Maggiore.