In this study, the effect of ammonia (NH3) as a disturbance on the microbial communities during anaerobic digestion (AD) was examined. NH3 was progressively added into 5 stabilized semi-continuous bio-reactors containing biowaste at 5 different NH3 loading rates until an inhibitory concentration was reached, and maintained at this concentration for 3 hydraulic retention times (75 days). The performance and the microbial community of the digesters were analyzed. The digesters performances were hindered at higher NH3 loading rates, marked by lower biogas productions and the accumulation of volatile fatty acids. At the microbial community level, changes depended on the NH3 loading rate. For instance, the digester with the highest NH3 loading rate showed a hydrogenotrophic methanogenic profile resulting from the growth of Methanosarcina spp. and acetate-consuming microorganisms, as well as the restricted growth of obligate acetoclastic archaea. This work can be helpful for the design of future acclimation strategies in AD.