Founded in 1889 and inaugurated in 1890, the museum's first aim was to collect and exhibit archaeologic materials unearthed during the excavations after the union of Rome with the Kingdom of Italy.

The initial core of its collection originated from the Kircherian Museum, archaeologic works assembled by the antiquarian and Jesuit priest, Athanasius Kircher, which previously had been housed within the Jesuit complex of Sant'Ignazio. The collection was appropriated by the state in 1874, after the suppression of the Society of Jesus. Renamed initially as the Royal Museum, the collection was intended to be moved to a Museo Tiberino (Tiberine Museum), which was never completed.

In 1901 the Italian state granted the National Roman Museum the recently acquired Collection Ludovisi as well as the important national collection of Ancient Sculpture. Findings during the urban renewal of the late 19th century added to the collections.

In 1913, a ministerial decree sanctioned the division of the collection of the Museo Kircheriano among all the different museums that had been established over the last decades, such as the National Roman Museum, the National Etruscan Museum of Villa Giulia and the Museum of Castel Sant'Angelo.

Its seat was established in the charterhouse designed and realised in the 16th century by Michelangelo within the Baths of Diocletian, which currently houses the epigraphic and the protohistoric sections of the modern museum, while the main collection of ancient art was moved to the nearby Palazzo Massimo alle Terme, acquired by the Italian state in 1981.

The reconversion of the area of the ancient bath/charterhouse into an exhibition space began on the occasion of the International Exhibition of Art of 1911; this effort was completed in the 1930s.