Studies on the antibacterial activities of the leaf extract of Citrullus colocynthis (Cucurbitaceae), a medicinal plant used for the treatment of various ailments was carried out using agar disc diffusion technique. Broad spectrum of an antimicrobial activity against sixteen bacteria and six fungal strains. No correlation was observed between susceptibility of test strains with plant extracts and antibiotic resistance behavior of the microbial strains. Qualitative phytochemical test of active extracts demonstrated the presence of phenols, tannins and flavonoids as active constituents. The significant antimicrobial activity of active extracts was compared with the standard piperacillin (100µg/disc) and Gentamicin (10µg/disc). The results obtained in the present study suggest that Citrullus colocynthis can be used in treating diseases caused by the test organisms.