Aims: The aim of this work was to use response surface methodology (RSM) approach, a statistical mathematical tool, to model effects and interactions of glucose oxidase (GOD), glucose, lactoperoxiclase (LPO) and pH-values on the thiocyanate (SCN-) peroxidation, to determine the best concentrations of lactoperoxiclase system (LP-s) components in order to obtain maximal SCN- peroxidation and so to enhance the LP-s antibacterial effects. Methods and Results: Experimental design using RSM was used for modelling effects and interactions of GOD (28.5-142.5 IU l(-1)), glucose (0.55-11.11 mmol l(-1)), LPO (0-6284 IU l(-1)) concentrations, and pH-values (6.0-7.4) on thiocyanate peroxidation. A fixed SCN-concentration of 0.5 mmol l(-1) was used. Experiments were carried out at 4 or at 25 degrees C in 0.1 mol l(-1) phosphate buffer. Optimized concentrations for both temperatures (4 and 25 degrees C) were quite similar and were 85.5 IU l(-1) for GOD, 8 mmol l(-1) for glucose and 3927.5 IU l(-1) for LPO at an initial pH-value of 6.5. SCN-peroxidation was more efficient at 25 than at 4 degrees C. At 4 degrees C, no interaction between factors occurred. At 25 degrees C, thiocyanate peroxidation was affected by GOD/glucose, GOD/pH and LPO/pH. Thiocyanate peroxidation was mainly increased by glucose and LPO factors. The optimized system had a bacteriostatic effect on Listeria monocytogenes CIP 82110(T) and a strong bactericidal effect on Pseudomonas fluorescens CIP 6913(T). Conclusions: Appropriate combinations of LPO, GOD, glucose concentrations and pH-values allowed maximal thiocyanate peroxidation and enhanced the antibacterial effect of the LP-s. Significance and Impact of the Study: This optimization by RSM approach allowed a better understanding of the LP-s functioning, the description of the component impacts on the SCN- peroxidation, and the observation of different interactions between the factors. The antimicrobial efficiency of LP-s can be enhanced by better concentration ratios of the LP-s components.