Peach fruit is one of the most economically widespread temperate fruits, whose productivity, and nutritional and sensory qualities are determined by interactions among several environmental and genetic factors, rootstocks, agronomic practices and pedo-climatic conditions. In recent years, climate change has prompted peach breeding programs to use specific rootstocks that are well adapted to unusual soil and climate characteristics, thus improving the plant’s adaptability and fruit quality. The aim of this work was to assess the biochemical and nutraceutical profile of two different peach cultivars, considering their growth on different rootstocks over three crop years. An analysis was carried out evaluating the interactive effect of all factors (i.e., cultivars, crop years and rootstocks) revealing the advantages or disadvantages on growth of the different rootstocks. Soluble solids content, titratable acidity, total polyphenols, total monomeric anthocyanins and antioxidant activity in fruit skin and pulp were analyzed. An analysis of variance was performed to assess the differences between the two cultivars considering the effect of rootstock (one way) and crop years, rootstocks and their interaction (two ways). In addition, two principal component analyses were performed separately on the phytochemical traits of the two cultivars to visualize the distributions of the five peach rootstocks during the three crop years. The results showed that fruit quality parameters are strongly dependent on cultivars, rootstocks and climatic conditions. All these aspects could be useful for the choice of rootstock in relation to agronomic management, making this study a valuable tool for choosing the best rootstock, considering simultaneously more factors affecting peaches’ biochemical and nutraceutical profile.