The consumer demand for an enhanced diet intake of antioxidants and bioactive compounds is continuously rising. This work aims to evaluate the fruit nutritional composition and antioxidant activity of five tomato germplasm varieties, alongside a commercial F1 hybrid. Three varieties bear small-fruit (14–40 g), while two varieties and the commercial cultivar yield large fruit (150–300 g). Genotypes under study were cultivated in a greenhouse under the same environmental conditions. Fat, protein, carbohydrate, total phenol, total flavonoid, lycopene, and ascorbic acid contents were assessed at two fruit maturity stages (breaker, red ripe). For both hydrophilic and lipophilic fractions, antioxidant behavior was also evaluated by employing DPPH and FRAP assays. Small-fruit varieties generally possess higher fat and ascorbic acid content, as well as hydrophilic FRAP values as compared to large-fruit ones. In all varieties, lycopene content and lipophilic fraction radical scavenging capacity was considerably higher at red ripe stage. At red ripe stage, all germplasm varieties were clearly and consistently superior in terms of antioxidant activity at the lipophilic fraction owing to enhanced lycopene content. The results emphasize the value of reintroducing germplasm varieties in breeding programs and suggest that local varieties generally encompass high quality features.