Medicinal plants are the important source of potentially useful chemotherapeutic agents which have made enormous contributions to human health and well-being. The methanolic extract of leaves of Eucalyptus tereticornis for antibacterial activity against the bacterial strain Escherichia coli was investigated. Response surface methodology (RSM) using central composite design (CCD) was adopted to optimize the effect of process variables.The optimum conditions for maximum antibacterial activity (zones of inhibition, mm) were found out to be E. tereticornis extract i.e., Limonene, 3 mL; 1, 8-Cineole, 11 mL; Terpinen-4-ol, 15 mL; pH, 7.0 and temperature, 40 C. The results suggest that the organic (methanol) extract of E. tereticornis could be a possible source to obtain new and effective herbal medicines to combat infections caused by multi-drug resistant microbial strains from community and hospital settings.