This study evaluates the synergistic antifungal effects between thyme essential oils and Lactobacillus plantarum cell-free supernatant (LCFS) against Penicillium spp. and in situ antifungal activity in rice grains. Thyme essential oil and LCFS showed remarkable antifungal activities against Penicil- Practical applicationsEssential oils are used as preservative in food industry and high concentration of essential oil is associated with negative organoleptic characteristics. This study presented a novel approach for synergistic antifungal effects by using the combination of thyme essential oil and Lactobacillus plantarum cell-free supernatant (LCFS) against Penicillium spp. and systematic evaluation of the antifungal effect by using fractional inhibitory concentration (FIC) index method. This approach will be a role model for future research on synergism and overcome the major drawbacks of using live bacteria and the negative effects arising from antimicrobial activities of essential oils.