Quaternary ammonium compounds have gained widespread attention due to their extensive enrichment in waste activated sludge (WAS) and potentially adverse effect to anaerobes. This study selected benzalkonium chlorides (BACs) as model to reveal the responses of anaerobic digestion of WAS to long-term stress of BACs. Results showed that the solubilization enhancement of WAS contributed by BACs was the acceleration of cell lysis, rather than the disruption of extracellular polymeric substances, and the accumulation improvement of short chain fatty acids (SCFAs) attributed to hydrolysis improvement and methanogenesis inhibition at either medium -or high level of BACs. In addition, a low level had no significant effect on the production of methane compared to control, with averages of 0.059 and 0.055 m3/(m3·d), respectively, whereas a medium level reduced methane production to 20% of control, and a high level almost completely inhibited methanogenesis. Correspondingly, BACs could shift microbial communities related to SCFAs and methane productions. For the bacterial community, a high level of BACs led to abundance reductions of Firmicutes, Bacteroidetes, Acidobacteria and Chloroflexi, but Synergistetes was increased to 10.5%, which was almost not detected either in control or at a low level of BACs. And for dominant archaeal community, they tended to be shifted from acetotrophic to hydrogenotrophic methanogens with BACs increasing from low to high level. These findings provided some new insights for the role of BACs in anaerobic digestion, as well as resource recovery from WAS.