Bacteriocins, antimicrobial compounds, display applications from food preservation to medicine. The aim of this study was the statistical optimization of a bacteriocin substance production from traditional cheese bacterium taxonomically affiliated as Lactobacillus pentosus TSHS. The effects of variables were first investigated using the Placket–Burman design. Central composite design was then used to compute incubation time and NaCl concentration optimum. Response surface methodology (RSM) revealed that maximum bacteriocin production was achieved at low NaCl concentration (0.13 mol/L) and an incubation time of 47 h. The validation experiments were implemented under optimal conditions with five replicates and the bacteriocin inhibitory activity was increased by three times. The research led to parameters that maximized pentocin activity, which can be expressed as a quadratic function of incubation time and NaCl concentration. The application of RSM resulted in an enhancement in pentocin production cost effective medium.