Mafai was born in Rome by Eleonora de Blasis and an unknown father. His surname, Mafai, was made up by the midwife. Little Mario grew up as an introverted and imaginative boy. He often wandered around the neighborhood and was captivated by paintings displayed in the windows of the antique dealers. As a young man, Mafai decided to follow his artistic vocation and started to attend the free nude painting classes at the Academy of Fine Arts. There he met Gino Bonichi, it became a lifelong friendship that would prove to be fundamental for both of them. Bonichi was a painter, called Scipione for his tall and imposing figure. At the academy, he also met the artist Antonietta Raphaël, who had just come from Paris and whom he would later marry. Together they spent weeks in the library of Palazzo Venezia, studying Italian and foreign masters: Velasquez, El Greco, Pontorno, Goya, and Piero della Francesca. They also discovered, albeit through very bad quality black and white reproductions, the paintings of the French impressionists, the Flemings, and the Germans.

In 1927, Mafai and Raphaël moved to a small apartment annex studio, which was frequented by Scipione and the sculptor Marino Mazzacurati. These gatherings gave rise to an association known as the Scuola Romana, which was sometimes called the Scuola di via Cavour, because the studio was on that street. In contrary to most of the older generation of artists, that had adopted a life of professorial, Mafai and the other Scuola Romana artists created a vigorous Bohemia scene. They were determined to paint only what they themselves felt deeply and resisted all attempts of the government to revive the classical cultural supremacy of Italy. During this period Mafai mostly painted views of the River Tiber in a deliberately unschooled manner, self-portraits and still-lifes, which showed the influence of Chaïm Soutine. His famous series of still-lifes of Fiori Secchi (dried flowers) are emblematic of the renewed form of painting by these young Roman artists. In response to the formal research of Giorgio Morandi, he emphasized the tonal quality of his paintings. The colors of the withered leaves and flowers in the open sunlight are bright but chaste, they punctuate the balanced composition and gives it a quiet sense of objectivity. This concentration on subtle gradations also endowed his still-lifes with a heightened, magical reality. In 1932, he made his debut at the XVIII Venice Biennale and his work became internationally known in traveling exhibitions in the United States.

In 1939, Mafai fled to Genoa for the sake of his wife, who was the daughter of a Lithuanian Rabbi, fearing increasingly for her safety in the anti-Semitic atmosphere of their nation. He started to make the series of Fantasia paintings, in which he used an expressionist aesthetic to voice a critique of Fascism. In these works, Mafai dismantled ideas about fascist (and Futurist) imagery, beauty, and the ideal body. In one painting, for example, he showed the grotesquely caricaturized naked bodies of the conquerors. In the fantasia series, but also in paintings depicting the demolitions of the suburbs of Rome, Mafai reflected on the brutal realities of war, in a similar manner as the famous engravings Disasters of War by Goya, and paintings of Gericault, and Grosz.

After the fall of the Fascist regime, the importance of Mafai’s work was widely recognized. Following the death of the artist in 1965, the IX Quadriennale di Roma celebrated his artistic career with a major retrospective, which affirmed Mafai’s position on the international stage. His work can be found in various major collections such as Galleria nazionale d'arte moderna e contemporanea, Rome and Museo d'arte moderna e contemporanea di Trento e Rovereto, Trento.

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