The objective of this study was to evaluate the effect of bioconversion conditions on reuterin production by Lactobacillus reuteri using response surface methodology (RSM). A central composite design consisted of three independent factors at 5 levels: concentration of Lactococcus reuteri (log CFU/ml), glycerol concentration (mM), and incubation time (h) was set up and consisted of 18 experimental runs. The dependant factor was the relative reuterin concentration expressed through its antibacterial activity (Units reuterin per ml, U/ml) against L. monocytogenes using microbroth dilution assay. A polynomial equation for prediction of reuterin production (U/ml) produced by L. reuteri was created based on obtained data. Results showed that incubation time, bacterial and glycerol concentrations had linear positive effects on reuterin production. The quadratic effect of incubation time had negative effect on reuterin production in which at a fixed bacterial concentration of L. reuteri, a longer incubation time with low concentration of glycerol will cause the reduction in relative reuterin concentration. It was found that approximately 450 U/ml can be produced by L. reuteri with glycerol concentration ranging from 320 to 340 mM mM, L. reuteri concentration of 9.4 log CFU/ml and 2 h of incubation. Results demonstrated that applying suitable conditions for glycerol bioconversion into reuterin by L. reuteri, a high relative reuterin concentration could be obtained and used for food preservation against L. monocytogenes.