Abstract This work presents antibacterial potential and anatomical investigation of Calotropis procera (Aiton) Dryand. Populations from different areas of Mirpur (Azad Jammu & Kashmir), Pakistan. In the research, three different areas (viz: Jarikus, Khaliqabad and Jatlan) were selected for population sampling (leaf parts) having no symptom of disease. Leaf powder was macerated in two solvents and tested against four pathogenic bacterial strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa). The methanolic extract depicted highest zone of inhibition (ZI) values with 14.67±1 mm against Pseudomonas aeruginosa (PA) while aqueous extract showed good ZI (14±1 mm) against this bacterium. Extract from Jatlan sample showed highest ZI values for methanolic fraction against Staphylococcus aureus and least effect against Bacillus subtilis (13±1 mm). Against E. coli methanol showed 14.7±0.8 mm and aqueous showed 11±0.5 mm zone of inhibition. Against Pseudomonas aeruginosa methanol showed 12±0.76 mm and aqueous showed 12.7±0.5 mm zone of inhibition. Among all three sampled areas, methanolic leaf extracts of Jatlan area showed very good ZI activity with 19±1 mm against Staphylococcus aureus followed by extracts of Khaliqabad samples with 14.67±1 mm against Escherichia coli. Among aqeous extracts leaves of Khaliqabad area presented highest antibacterial potential (14±1 mm) agaisnt Pseudomonas aeruginosa. It was observed that maximum zone of inhibitions were showed by methanolic extracts as compare to aqueous extracts. The maximum zone of inhibition in methanolic extracts of Jatlan was 18.7 mm among all areas. An anatomical analysis was made to explore leaf phenetics and its impact on concentration of phytochemicals with subsequent variation analysis in antibacterial potential. It was found that plant leaf samples of Khaliqabad had highest stomatal index (SI) 538 and same was found best antibacterial activity against various strains of bacteria. SI of Jatlan 408.7 followed by samples of Jarikus with SI values of 242.8. It was confirmed that there is direct and positive correlationship between SI and ZI of leaves of plants collected from different localities. These findings depict that leaves of Calotropis procera (Aiton) Dryand have good antibacterial activity confirming the past ethnomedicinal folklore applications of the plant and this result findings can be used as source of novel drug development from such herbal therapeutic products.