Girolamo Macchietti trained in Florence. He worked under Vasari between 1555 and 1561, redecorating the Palazzo Vecchio where he was employed as a designer of tapestries. Macchietti then studied in Rome for a period of around two years, before returning to Florence and becoming a member of the newly founded Accademia del Disegno.
Macchietti’s painting shows the influence of Vasari's elaborate mannerist style and draws on Parmigianino and Raphael. He also produced less elaborate and more naturalistic works, possibly influenced by his time in Rome. He contributed two sophisticated paintings – the 'Baths of Pozzuoli' and 'Medea and Jason' (c.1570–1572) – to the decorative scheme of Francesco I de' Medici's 'studiolo' in the Palazzo Vecchio.
Macchietti died in 1592, spending his final years in Naples, Benevento and Spain. His later works were more schematic, lacking the extravagant qualities of some of his earlier work.