Benzalkonium chloride (BAC) is a commonly used disinfectant in dairy production. To investigate the effects BAC has on lactic acid bacteria (LAB) in fermented milk, the mechanisms by which BAC influences LAB gene functions and metabolic pathways were investigated using metagenomic analysis. The results identified a total of 216 LAB taxa isolated from fermented milk samples containing three different concentration levels of BAC. Streptococcus thermophilus was identified as the predominant species, accounting for 99.78%~98.45% of the total bacterial strains. BAC significantly inhibited the growth and proliferation of LAB. Gene function annotation analysis showed that 16,112 genes were annotated in the COG database, with the highest impact observed on amino acid transport and metabolic functional units. In the KEGG database, carbohydrate and amino acid metabolism were the primary pathways involved, with gene counts decreasing by 7.76%~9.10% as the BAC concentration increased. CAZy analysis indicated that glycoside hydrolases and glycosyl transferases were the most abundant enzyme families (accounting for 79.08%~80.38%), and BAC significantly upregulated genes in the GT8 family. Differential metabolic pathway analysis demonstrated that BAC downregulated glucose kinase and pyruvate kinase. This resulted in a decreased acidification capacity in the BAC2.0 sample group, potentially affecting the texture and flavor characteristics of fermented milk. These findings provide critical insights into the potential impact that BAC has on LAB and lay a foundation for further exploration of BAC-related risks in other fermented foods.