This investigation reports the variability of antioxidant components and antioxidant activities of six watermelon cultivars (cvs) (four commercial cvs Aramis, Crimson Sweet, Dumara, Giza, and two new selections P503 and P403 produced by the National Agricultural Research Institute of Tunisia) as influenced by sampling area. All cvs were simultaneously grown in an open-field and subjected to identical horticultural practices in order to minimize the effects of environmental conditions and maximize those related to genotype. Significant differences were found between watermelon cvs for lycopene, phenolics, flavonoids, ascorbic acid (AsA), dehydroascorbic acid (DHA) and total vitamin C (AsA + DHA) contents, as well as in the antioxidant activity of their hydrophilic and lipophilic fractions. P503 cv showed the highest lycopene and flavonoid contents. Crimson Sweet and Giza cvs showed the highest HAA and LAA when TEAC was used as assay method, while the highest HAA and LAA were detected in Giza and Dumara cvs and in P503 cv, respectively, when FRAP assay was used. This study demonstrates that the amount of each specific antioxidant, as well as the HAA and LAA, were both influenced by genotype and sampling area, emphasizing the need to evaluate watermelon biodiversity in order to improve its nutritional value.