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Objective:The column chromatographic fractions of chloroform (CH1, CH2, CH3) seed extracts of Argemone mexicana(A. mexicana) were screened for antibacterial activity and phytochemical analysis. Methods:CH3 fraction was isolated and identified by column chromatography, thin layer chromatography, spectral data analysis and phytochemical screening were used for analysis. Results: CH3 fraction was significantly active at 4 to 64 mg/mL against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa with minimum inhibitory concentration of 1.5625 to 3.125 mg/mL. The active fraction (CH3) revealed presence of alkaloid with retention factor value of 0.44. The active antibacterial agent in the most potent fraction (CH3) was isolated and identified as N-demethyloxysanguinarine by thin layer chromatography (TLC) and phytochemical screening. The CH1 and CH2 fractions did not show inhibitory activity. Conclusions:The results support the ethnomedicinal use of seeds of A. mexicana for treatment of bacterial diseases.