To isolate and analyze bacteria with wilt-resistant properties from the fermentation residue of kitchen wastes, as well as explore their potential for new applications of the residue. A total of six bacterial strains exhibiting wilt-resistant capabilities were isolated from the biogas residue of kitchen waste fermentation. Using a polyphasic approach, strain ZL6, which displayed the highest antagonistic activity against cotton wilt, was identified as belonging to the . Bioassay results demonstrated that this strain possessed robust antagonistic abilities, effectively inhibiting spore germination and mycelial growth. Furthermore, ZL6 exhibited high temperature resistance (42°C), nitrogen fixation, and phosphorus removal activities. Pot experiments revealed that ZL6 fermentation broth treatment achieved a 47.72% biological control effect compared to the control group. Through activity tracking and protein mass spectrometry identification, a neutral metalloproteinase () was hypothesized as the main virulence factor. The mutant strain ZL6Δ exhibited a significant reduction in its ability to inhibit cotton wilt compared to the strain ZL6. While the inhibitory activities could be partially restored by a complementation of gene in the mutant strain ZL6CMΔ. This research provides a theoretical foundation for the future development and application of biogas residue as biocontrol agents against wilt and as biological preservatives for agricultural products. Additionally, this study presents a novel approach for mitigating the substantial amount of biogas residue generated from kitchen waste fermentation.