Little is known of Castagno's early life, and it is also difficult to ascertain the stages of his artistic development owing to the loss of many of his paintings and to the scarcity of documents regarding his extant works. As a youth, he was precocious. He executed a mural of Cosimo de' Medici's adversaries (rebels hanging by their heels) at the Palazzo del Podestà in Florence, earning himself the byname Andreino degli Impiccati (“Little Andrea of the Hanged Men”). It is known that he went to Venice in 1442, and frescoes in the chapel of Saint Tarasio in San Zaccaria are signed and dated by both him and Francesco da Faenza.

His first notable works were a Last Supper and three scenes from the Passion of Christ—a Crucifixion, a Deposition, and a Resurrection—all executed in 1447 for the refectory of the former Convent of Sant'Apollonia in Florence, now known as the Cenacolo di Sant'Apollonia. These monumental frescoes, revealing the influence of Masaccio's pictorial illusionism and Castagno's own use of scientific perspective, received wide acclaim.

In 1451 Castagno continued the frescoes at San Egidio begun earlier by Domenico Veneziano. The light tones that Castagno adopted for his outstanding St. Julian (1454–55) show Domenico's influence.

In a work for a loggia of the Villa Carducci Pandalfini at Legnaia, Castagno broke with earlier styles and painted a larger-than-life–size series of Famous Men and Women, within a painted frame. In this work, Castagno displayed more than mere craftsmanship; he portrayed movement of body and facial expression, creating dramatic tension. Castagno set the figures in painted architectural niches, thus giving the impression that they are actual sculptural forms. He achieved similar force in his David with the Head of Goliath (c. 1450–55), painted on a shield. His last dated work (Florence Cathedral) is an equestrian portrait of Niccolò da Tolentino. Castagno's emotionally expressive realism was strongly influenced by Donatello, Domenico, and perhaps Piero della Francesca, and Castagno's work in turn influenced succeeding generations of Florentine painters, including Antonio del Pollaiuolo (Pollaiuolo brothers) and Sandro Botticelli (Botticelli, Sandro).