The aim of this study was to evaluate the effect of inoculum to substrate radio (ISR) on biomethane potential of chicken manure using cattle slurry as inoculum. Biomethane potential assays were carried out at 39 °C mesophilic temperature. Total fatty acids, total alkalinity, ammonium concentration and accumulative methane volume were measured to evaluate organic load (16.6, 11.0, 8.3, 6.6 y 5.5 g VS/L). The highest biomethane potential (0.58 m3 CH4/kg SV) was reached when anaerobic biodegradation was carried out to ISR of 1.0. The results demonstrated that chicken manure is a potential substrate to be degraded by anaerobic digestion and process performance is directly proportional to substrate concentration. This study also confirms that ISR allow dilution of inhibitory components as ammonium by the case of chicken manure.