This study evaluated the fruit by-products (peels, seeds, and fibrous strands) from 11 pumpkin genotypes cultivated in Greece aiming to valorize them as natural sources of bioactive compounds. Five compounds, including (-)-epicatechin and chicoric acid isomers, were identified in peels, while seeds and fibrous strands mainly contained (-)-epicatechin. Organic acids and tocopherols varied significantly among genotypes, with oxalic, quinic, and malic acids being predominant. Total tocopherols content (mg/100 g) ranged up to 7.38 ± 0.03 in fibrous strands, 30.7 ± 0.2 in peels, and 14.58 ± 0.09 in seeds. Extracts exhibited potent antioxidant activity, particularly the seeds of genotypes "V5" and "V6", and strong antimicrobial effects, notably the peels of "V2 T" and "V11", which showed significant inhibition of microbial strains. These findings contribute to the advancement of sustainable practices in the agro-industrial waste management, as well as to the production of functional natural ingredients for various industrial applications.