A designated bioactive peptide “Hispidalin” purified from the seeds of Benincasa hispida, which is a medicinal plant, belongs to Cucurbitaceae family. Purification was achieved by using a procedure consisting of extraction from potassium phosphate buffer followed by FPLC and HPLC steps. Based on amino acid residue, this peptide is amphipathic and basic with one net positive charge having isoelectric pH 8.1. This peptide is without sulphur containing amino acid suggesting its extended conformation lacking double bond secondary structure. The results obtained from MALDI-TOF suggested that Hispidalin is of molecular mass 5.7 KDa with 49 amino acid residues and confirmed SDS-PAGE resolved ∼6.0 KDa protein band. This novel and unknown peptide “Hispidalin” showed broad and potent inhibitory effects against various human bacterial and fungal pathogens; its growth inhibition was significantly comparable with commercial antibacterial and antifungal drugs. The Hispidalin at 40 μg/mL concentration exhibited 70.8% DPPH free radical-scavenging activity and 69.5% lipid peroxide inhibition. Thus, in the present study, Hispidalin demonstrated remarkable antimicrobial and antioxidant potentials from the seeds of B. hispida.