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In this study, a newly isolated <i>Pediococcus acidilactici</i> F3 was used as probiotic starter for producing fermented soymilk to enhance antioxidant properties with high antimicrobial activity against food-borne pathogens. The objectives of this study were to investigate optimized fermentation parameters of soymilk for enhancing antioxidant property by <i>P. acidilactici</i> F3 and to assess the dynamic antimicrobial activity of the fermented soymilk during co-culturing against candidate food-borne pathogens. Based on central composite design (CCD) methodology, the maximum predicted percentage of antioxidant activity was 78.9% DPPH inhibition. After model validation by a 2D contour plot, more suitable optimum parameters were adjusted to be 2% (<i>v</i>/<i>v</i>) inoculum and 2.5 g/L glucose incubated at 30 °C for 18 h. These parameters could provide the comparable maximum percentage of antioxidant activity at 74.5 ± 1.2% DPPH inhibition, which was up to a 23% increase compared to that of non-fermented soymilk. During 20 days of storage at 4 °C, antioxidant activities and viable cells of the fermented soymilk were stable while phenolic and organic contents were slightly increased. Interestingly, the fermented soymilk completely inhibited food-borne pathogens, <i>Salmonella</i> Typhimurium ATCC 13311, and <i>Escherichia coli</i> ATCC 25922 during the co-culture incubation. Results showed that the soymilk fermented by <i>P. acidilactici</i> F3 may be one of the alternative functional foods enriched in probiotics, and the antioxidation and antimicrobial activities may retain nutritional values and provide health benefits to consumers with high confidence.