Ammonia accumulation in biogas plants reactors is becoming more frequently encountered, resulting in reduced methane (CH4) production. Ammonia toxicity occurs when N-rich substrates represent a significant part of the biogas plant’s feedstock. The aim of this study was to develop an estimation method for the effect of ammonia toxicity on the CH4 production of biogas plants. Two periods where a biogas plant operated at 3200 mg·L−1 (1st period) and 4400 mg·L−1 (2nd period) of ammonium nitrogen (NH4+–N) were examined. Biomethane potentials (BMPs) of the individual substrates collected during these periods and of the mixture of substrates with the weight ratio used by the biogas plant under different ammonia levels (2000–5200 mg·L−1 NH4+–N) were determined. CH4 production calculated from the substrates’ BMPs and the quantities used of each substrate by the biogas plant was compared with actual CH4 production on-site. Biogas plant’s CH4 production was 9.9% lower in the 1st and 20.3% in the 2nd period in comparison with the BMP calculated CH4 production, of which 3% and 14% was due to ammonia toxicity, respectively. BMPs of the mixtures showed that the actual CH4 reduction rate of the biogas plant could be approximately estimated by the ammonia concentrations levels.