Herbs are natural remedies for various diseases with higher safety and efficacy profile. India is gifted with varieties of large number of medicinal herbs because of variety of climatic conditions and seasons favourable for growth of many species of plants. Amongst the large number of herbal drugs existing in India, very few have been studied systematically so far. Citrullus vulgaris is a highly valuable drug and is one of the essential ingredients in the most of the compound preparations included in Ayurvedic literature. It is well known for number of pharmacological activities like hepatoprotective activity, anti-inflammatory, wound healing, Antidibetic activity, hypotensive effect, immunomodulating property, bronchial catarrh, dysentery, diarrhea and to prevent falling of hair, promotes the growth of hair, and antimicrobial activity against both gram-positive and gram- negative bacteria. Its fruit juice active constituents shows antiseptic, insecticidal and parasiticidal properties, against conjunctivitis and is used also to check hemorrhage from cuts, bruises and wounds insect repellent. It is also used as bioadsorbent for chromium. Here we try to make attempt for focusing on antimicrobial activity of Citrullus seeds extract. Their antibacterial activities were evaluated in vitro against clinical bacterial isolates. In effort to identify novel bacterial agents, this study was initiated to evaluate the antimicrobial properties of Citrullus seed extract against E. coli and Staphylococcus aureus by using Agar well-diffusion assay. The seed extracts demonstrated good antimicrobial activity against these bacteria tested with inhibition zones. The minimal inhibitory concentration (MIC) values of extract against the tested bacteria were found to 47.3 mg/ml and 52.20 against E.coli and Staphylococcus aureus respectively. The methanolic extract of seeds exhibited a pronounced activity against E. coli and Staphylococcus aureus. The minimum inhibitory concentration was found in methanolic Extracts of Citrullus vulgaris against test organisms