Founded in 1288 by Folco Portinari, the father of the Beatrice beloved by Dante Alighieri, the hospital represents an early and efficacious example of health care in Italy and in Europe. Under the Medicean governments it was expanded and developed significantly. The Hospital of Santa Maria Nuova was administered by rectors, known as "spedalinghi". On November 17, 1783, through a grand-ducal decree, Peter Leopold of Lorraine approved its hospital rules, the first officially introduced in Tuscany, of political and financial nature, designed to curb excessive expenditure and correct irregularities in management. The main artificers were the grand-ducal commissary Marco Covoni and Vincenzo Chiarugi, who conducted a thorough verification of the situation in the hospitals and charity institutions of the Grand Duchy.

Since the 15th century a garden where medicinal plants were grown had been annexed to the Hospital. The chief function of this "giardino dei semplici" was that of furnishing herbs to the Spezieria, or pharmacy, which had also existed at the Hospital since the 15th century. The first catalogue of plants from the garden, compiled by the director Giuseppe Baldi, dates from the middle of the 17th century. In the early 18th century, thanks to the activity of Sebastiano Franchi, Niccolò Gualtieri and Gaetano Moniglia, the garden developed significantly, also through the contribution of Abbot Giovanni Lapi from Borgo San Lorenzo, "reader" in Botany at the School of Medicine and Pharmacology. Near the end of the century the garden entered a slow decline, in spite of the management of Antonio Targioni Tozzetti and his son Adolfo Targioni Tozzetti, who directed it up to 1860. In 1881 it was definitively suppressed.

In 1688 a "spedaletto" was constructed in the Hospital, destined to serve as "Pazzeria", or madhouse, in which were hospitalised male patients too poor to pay the charges for accommodation in the nearby hospital of Santa Dorotea, known as "the Madmen's" hospital. Starting in 172, women too were accepted in the Pazzeria, but in 1780, with the opening of specialised departments, the mentally ill patients were all transferred to the Boniface Hospital.

Closely linked to the Hospital's history is that of the annexed School of Surgery. It accepted young men training to become physicians, surgeons or pharmacists. In this hospital school the teaching was essentially practical, with lessons taught directly at the bedside of patients and observations on cadavers. In 1785 the school was enriched by a precious group of surgical instruments donated by Peter Leopold. Subsequently, it experienced a flourishing period thanks to the work of Paolo Mascagni. With the restoration of the Lorraines, in 1840, what was to become the School of Completion and Perfecting in Practical Medical-Surgical Studies at the Hospital of Santa Maria Nuova was established in definitive form. Its activity ceased in 1859 when the Casati law incorporated it in the Institute of Higher Studies and Perfection of the Kingdom of Italy. Among the many subjects taught at the School of Surgery, Pathological Anatomy, Mental Disorders and Obstetrics were especially important.

In Florence Obstetrics, originally taught by the so-called "Women's surgeons", had its first chair in 1756 when Grand Duke Francis Stephen appointed Giuseppe Vespa "maestro di grembiule" (master of the apron) at the Hospital of Santa Maria Nuova and delivery room director. Starting in 1773, a delivery room for the poor was set up in the Hospital destined by Peter Leopold to practical instruction for the midwives of the Grand Duchy. In 1783 the Clinic of Obstetrics took the place of the old school of obstetrical instruction located at the Hospital of Orbatello. Starting from 1806 Giuseppe Galletti directed the practical instruction of surgeons and midwives, utilising wax models for educational purposes. In the following years, separation between practical and theoretical teaching was retained; consequently, while practical exercises took place at the Hospital of the Innocent, theoretical lessons were taught at Santa Maria Nuova.