A 3-year (2011–2013) field trial was carried out in a mature vineyard (Vitis vinifera L., cv. Sangiovese), planted in 2003, to assess physiological responses of grapevines to biodynamic management. Starting in 2007, the vineyard was managed with organic production protocols in accordance with EC Regulations (834/2007). In 2008, the vineyard (2 ha) was divided in two large plots, with each plot having similar soil physico-chemical properties. One of the plots was managed with organic protocols per EC Regulations and the other with biodynamic practices, consisting of spray application of preparations 500, 500 K, fladen and 501. During the 2011–2013 season, the biodynamic preparations were used at least twice per year, with the exception of 501 that was applied only once in 2013. Concentration of hormones and mineral elements in biodynamic preparations were determined. Biodynamically managed vines showed lower stomatal conductance in all years and lower leaf water potential in 2012. Leaf photosynthetic activity was not influenced by cultivation method. Biodynamic management led to an increase in leaf enzymatic activities of endochitinase (EC 184.108.40.206), exochitinase (β-N-acetylhexosaminidase, EC 220.127.116.11 and chitin 1,4-β-chitobiosidase) and β-1,3-glucanase (EC 18.104.22.168), which are typically correlated with plant biotic and abiotic stresses and associated with induced plant resistance. Year effects were observed with 1,3-β-glucanase, whose activity in 2012 was 4.1-fold higher than in 2013. Disease incidence and grape yields were not different between organic and biodynamic treatments. This study provided a strong indication of a stimulation of natural defense compounds in grapes grown under biodynamic cultivation, but subsequent effects on plant protection and productivity require further evaluation.