Response surface methodology (RSM) is frequently used for optimization studies. In the present work, RSM was used to determine the antimicrobial activitiesof grapefruit seed extract (GFSE) and a lactic acid mixture (LA) against Staphylococcus aureus, Bacillus cereus, Escherichia coli, Salmonella typhimurium, Pseudomonas fluorescens, and Vibrio parahaemolyticus. A central composite design was used to investigate the effects of independent variables on dependent parameters. One set of antimicrobial preparations included mixtures of 1% (w/w) GFSE and 10% (w/w) LA, in which the relative proportions of component antimicrobials varied between 0 and 100%. In further experiments, the relative proportions were between 20% and 100%. Antimicrobial effects against various microorganisms were mathematically encoded for analysis. The codes are given in parentheses after the bacterial names, and were S. aureus (), B. cereus (), E. coli (), S. typhimurium (), P. fluorescens (), and V. parahaemolyticus (). The optimum antimicrobial activity of the 1% (w/w) GFSE:10% (w/w) LA mixture against each microorganism was obtained by superimposing contour plots ofantimicrobial activities on measures of response obtained under various conditions. The optimum rangesfor maximum antimicrobial activity of a mixture with a ratio of 1:10 (by weight) GFSE and LA were 35.73:64.27 and 56.58:43.42 (v/v), and the optimum mixture ratio was 51.70-100%. Under the tested conditions (a ratio of 1% [w/w] GFSE to 10% [w/w] LA of 40:60, and a concentration of 1% [w/w] GFSE and 10% [w/w] LA, 70% of the highest value tested), and within optimum antimicrobial activity ranges, the antimicrobial activities of the 1% (w/w) GFSE:10% (w/w) LA mixture against S. aureus (), B. cereus (), E. coli (), S. typhimurium (), P. fluorescens (), and V. parahaemolyticus () were 24.55, 25.22, 20.20, 22.49, 23.89, and 28.04 mm, respectively. The predicted values at optimum conditions were similar to experimental values.