The Gallery was founded on 10 April 1900 by Law ΒΨΛΔ΄, which provided for a supervisor; the painter Georgios Iakovidis (1853-1932) was appointed to this post. On June 28 1900, the Gallery’s rules of operation were laid down, according to which the artworks in its collection were divided into six sections, including ‘Byzantine… Medieval… Modern art (oil paintings)… watercolours, pastels and drawings… copies of paintings in other collections, and prints (woodcuts, copper engravings and other kinds of engravings…)’. The collection core consisted of 258 works of art from the collections of the National Technical University (NTUA) and the University of Athens, which were delivered to Georgios Iakovidis by NTUA’s director, Anastassios Theofilas, on 28 July 1900, as well as by the collection of lawyer and art connoisseur Alexandros Soutsos (1839-1895), who bequeathed his estate to the Greek state for the purpose of establishing and maintaining a museum of painting. His collection was delivered by Natalia Soutsou on 21 June 1901.

On 24 February 1906, a royal decree was issued, whereby a six-member supervisory committee was appointed, consisting of leading personalities of the country’s political and entrepreneurial life: the later prime minister, Stefanos Skouloudis, and Stefanos P. Rallis, Markos Dragoumis, Petros Kalligas, Spyridon Stais and ThemosAnninos. The committee, in co-operation with the supervisor, was responsible for displaying, enriching, developing and preserving the gallery’s collections. In addition, the Gallery secured financial support – 3,000 Drachmas per year – for the first time, from the bequest of the Greek-diaspora benefactor, Dimitrios Doridis, who had left his fortune to the Greek nation to be used in public institutions.

Collection enrichment was facilitated by adopting laws that ensured the allocation of art works to the Gallery, as well as by generous contributions by individuals inside and outside Greece. In this context, on 6 April 1911, the Georgios Averoff Estate Trustees donated to the Gallery the 80 paintings in Averoff’s collection, as well as 15 more, acquired with the estate’s funds, all of which went on display in a room of their own, named ‘Averoff Art Gallery’, according to the donor's wish.

The first major exhibition of works from the Gallery’s collection was announced in 1912, but only opened in 1915. Some 300 paintings, conserved by the painter Georgios Hatzopoulos, were shown in six large NTUA rooms.

In collaboration with the supervisory committee, Georgios Iakovidis also made strenuous efforts to resolve the housing issue, as the Gallery was in need of housing of its own, having been housed since its founding on the upper floor of NTUA’s main building. These efforts fell through due to disagreement over the building’s architectural style and location. In 1914, by law 477, a plot of land on Vassilissis Sofias Avenue and Rizari Street had been allocated; however, the building was not constructed, due to the objections of the military, to which the site belonged.

Nevertheless, in order to further the Gallery’s educational programme, Iakovidis introduced free admission for the public every Saturday and Sunday to NTUA’s rooms, which also served as an exhibition venue. He also published the Gallery’s first catalogues, in 1906 (which included the rules of operation) and 1915; moreover, he succeeded in securing two positions for ‘curators’, as conservators were called at the time, taken up by the painters Georgios Hatzopoulos and Odysseas Fokas.