The present study was carried out to investigate the antibacterial and antifungal potential of Citrullus lanatus seeds. Determination of antimicrobial activity of methanol extract of Citrullus lanatus seed was carried out against 10 bacterial species (Staphylococcus aureus, Klebsiella pneumoniae, Bacillus subtilis, Escherichia coli, Salmonella typhi, Enterococcus faecalis, Vibrio cholerae, Shigella dysenteriae, Proteus mirabilis, Pseudomonas aeruginosa) and 5 fungal species (Aspergillus flavus, Aspergillus niger, Penicillium notatum, Trichophyton mentagrophytes, Candida albicans). Inhibition zones formed by the extract were compared with the standards: Streptomycin and Amphotericin B. The methanolic seed extract at a concentration of 1000 μg/ml was effective against bacteria: Vibrio cholerae (7 mm), Proteus mirabilis (7 mm), Shigella dysenteriae (7 mm), Staphylococcus aureus (6 mm), Escherichia coli (6 mm), Enterococcus faecalis (6 mm), B. subtilis (6 mm), Salmonella typhi (5 mm) and fungi: Aspergillus niger (13 mm), Aspergillus flavus (12 mm), Penicillium notatum (11 mm), Candida albicans (9 mm). The results of Citrullus lanatus seed extract demonstrated antimicrobial activity against the organisms tested. Hence, this extract can be used to discover novel bioactive natural products that may lead to the development of new antimicrobial drug.