The outbreak of epidemics such as African swine fever has intensified the use of disinfectants in pig farms, resulting in an increasing residual concentration of disinfectants in environmental media; however, the high-frequency excessive use of disinfectants that damage pig farm manure anaerobic fermentation systems and their mechanisms has not attracted enough attention. Especially, the complex effects of residual disinfectants on anaerobic fermentation systems for pig manure remain poorly understood, thus impeding the application of disinfectants in practical anaerobic fermentation systems. Herein, we explored the effects of glutaraldehyde disinfectant on methane production, effluent physicochemical indices, and microbial communities in a fully automated methanogenic potential test system (AMPTSII). The results show that adding glutaraldehyde led to remarkable alterations in methane production, chemical oxygen demand (COD), volatile solids (VS), and polysaccharide and phosphorus concentrations. During the anaerobic process, the production of methane displayed a notable decrease of 5.0–98% in all glutaraldehyde treatments, and the trend was especially apparent for treatments containing high levels of glutaraldehyde. Comparisons of the effluent quality showed that in the presence of 0.002–0.04% glutaraldehyde, the COD and total phosphorus (TP) increased by 12–310% and 15–27%, respectively. Moreover, the addition of 0.01–0.08% glutaraldehyde decreased the ammonium (NH4+-N) concentration and VS degradation rate by 7.7–15% and 4.9–26.2%. Furthermore, microbiological analysis showed that the glutaraldehyde treatments had adverse effects on the microbial community. Notably, certain functional bacteria were restrained, as highlighted by the decreases in relative abundance and microbial diversity by 1.3–17% and 0.06–21%, respectively. This study provides a theoretical basis for the rational use of disinfectants in anaerobic fermentation systems.