Bacteriocinogenic Lactobacillus fermentum BZ532 with novel bacteriocin LF-BZ532 was originally isolated from Chinese cereal fermented drink, showing an antimicrobial characteristic during fermentation. This study aimed to explore the in situ antimicrobial activities of L. fermentum BZ532 and co-culturing investigation against key food pathogens, i.e., Staphylococcus aureus and Escherichia coli K-12, was conducted during fresh bozai production. The growth of spoilage bacteria was suppressed and bacterial count was reduced to a significantly low level during 48 h of co-cultures. In situ production of antimicrobial compounds expressed positive activity against S. aureus and E. coli K-12, but negative acitivity against Salmonella sp. D104. The total viable count of bozai BZ-Lf (bozai fermented with BZ532 strain) had a comparatively lower viable count than bozai BZ-C (bozai as an experimental control without BZ532) during storage of 7 days. Titratable acidity of bozai treatments (BZ-C, BZ-Lf) was increased, while pH declined accordingly during storage of 7 days. The organoleptic quality of bozai BZ-C had low sensorial scores as compared with BZ-Lf during storage. In comparison with naturally fermented bozai (BZ-C), L. fermentum BZ532 (BZ-Lf) could significantly reduce the microbial spoilage and extend the shelf-life based on microbiological examination. Conclusively, L. fermentum BZ532 can be used as a bio-protective culture for improving the safety of bozai.