Thirteen Prunus rootstocks with different genetic background [five peach-almond hybrids: Adafuel, Adarcias, GF 677, PADAC 9902-01 and Rootpac 70; one P. persica x P. davidiana hybrid: Cadaman; three hexaploid P. insititia plums: Adesoto 101, PM 105 AD, and PM 150 AD; three plum x peach-almond hybrids (PADAC 04-01, PADAC 04-03 and PADAC 99-05), and a plum-almond hybrid (Rootpac R)] were budded with the ‘Big Top’ nectarine and evaluated in typical Mediterranean growing conditions. Biochemical fruit compounds, such as sugars and organic acids profile, and antioxidant compounds were assessed to enhance fruit quality and beneficial effects of ‘Big Top’. For three consecutive years (2014–2016), sugars and organic acids were analysed by high-performance liquid chromatography and phenolic compounds by spectrophotometry. Statistical analyses showed that rootstocks affected strongly fruit phytochemical composition, although it was also influenced by year. Compared with peach-based rootstocks, the hexaploid ‘Pollizo’ plums Adesoto 101 and PM 150 AD can increase the sweetness of the low-acid ‘Big Top’ nectarine, inducing higher contents of sugars and organic acids. Similarly, beneficial antioxidant effects can be enhanced on diploid plum based hybrids PADAC 04-01, PADAC 99-05 and Rootpac R, inducing higher contents on sugars and bioactive compounds (total phenols, flavonoids and vitamin C) and antioxidant capacities. High significant correlations were found between all studied compounds. New plum based rootstocks seem to induce higher fruit quality based on sugars and organic acids profile, and antioxidants compounds.