Three-compartment septic tanks are a prominently advocated environmentally sustainable sanitation facility in rural China. However, the comprehensive elimination efficacy and underlying mechanisms of pathogenic bacteria within septic tanks remain incompletely understood. In particular, the operational performance in low-temperature conditions has received limited attention in the existing literature. In this work, a simulation of the three-compartment septic tank treatment system was conducted under low-temperature conditions (15 °C). The operational results exemplify the synergistic interplay of volatile fatty acids (VFAs), NH₃-N, and bacterial communities, culminating in a partial reduction in Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, and Salmonella enteritidis, within the three-compartment septic tank. Their respective population abundances were decreased by magnitudes of 2.2, 1.3, 0.03, and 1.46 logarithmic units (copies/mL), respectively. Through the utilization of qPCR and physicochemical indicators, it was observed that the bactericidal effect of VFA primarily occurred during the initial 0–21-day period, while NH₃-N consistently proved to be the most vital sterilizing agent throughout the operation of the three-compartment septic tank. Predominant bacterial communities within the septic tank, such as Christensenellaceae_R-7_group, Brevundimonas, Acinetobacter, and Saccharimonadales, exerted substantial inhibitory impacts on Enterococcus faecalis, Escherichia coli, and Salmonella enteritidis through niche competition and suppression. In essence, this study elucidated the actual efficiency of elimination and the underlying mechanisms of typical pathogenic bacteria within three-compartment septic tanks in low-temperature conditions, thereby providing compelling evidence supporting the viability of environmentally sound treatment using such septic tanks. Concurrently, it also shed light on several limitations associated with this treatment approach, aiming to contribute valuable insights for the assessment of ecological risks and health hazards associated with the environmentally benign treatment of rural toilet waste.