Objective This study aimed to evaluate the antifungal efficacy and mechanism of Escherichia coli (E. coli) ΔtbpA mutant strain, providing experimental support for its use as a novel antifungal treatment strategy. Methods E. coli ΔtbpA was cultured, and its cell-free supernatant (CFS) was collected and filtered. Effect of CFS on Candida was analyzed through time-growth curve. The proliferation of Candida during co-culture with E. coli ΔtbpA was detected by plate counting. The adhesion ability of E. coli ΔtbpA and its tolerance to artificial gastric and intestinal fluids were detected, and its probiotic properties were explored. Its in vivo antifungal effect was evaluated by Caenorhabditis elegans-Candida albicans (C. albicans) infection model. The antifungal active ingredients in CFS were explored through Transwell chamber assay as well as enzyme/heat treatment experiments.Results The time-growth curve and co-culture experiment results indicated that E. coli ΔtbpA could significantly inhibit the proliferation of Candida. The survival time of C. albicans-infected Caenorhabditis prolonged significantly after feeding with E. coli ΔtbpA. E. coli ΔtbpA could tolerate artificial gastrointestinal fluid environment. Transwell chamber assay and enzyme/heat treatment results showed that its antifungal effect depended on the secreted protease-sensitive and heat-resistant substances. Conclusion E. coli ΔtbpA mutant strain inhibits Candida proliferation by secreting protein-like active substances, and has good adhesion ability and tolerance to artificial gastrointestinal fluid. It is expected to be used as a probiotic for the treatment of Candida infection.