This study was undertaken to optimize the conditions for the extraction of antibacterial activity of Perilla frutescens var. acuta leaf against Pseudomonas aeruginosa KCTC 2004 using the evolutionary operation-factorial (EVOP) design technique. Increased antibacterial activity was achieved at higher extraction temperatures and with a longer extraction time. Antibacterial activity was not affected by differing ethanol concentration in the extraction solvent. The maximum antibacterial activity of ethanolic extract of P. frutescens var. acuta leaf against P. aeruginosa, determined by the EVOP factorial technique, was obtained at an extraction temperature of 80 degrees C (R = -0.800**), 26 h (R = -0.731**) extraction time, and 50% (R = -0.075) ethanol concentration. The population of P. aeruginosa also decreased from 6.660 log CFU/mL in the initial set to 4.060 log CFU/mL in the third set. Also, scanning electron microscopy study of the ethanolic extract of P. frutescens var. acuta revealed potential detrimental effects on the morphology of P. aeruginosa.