The objective of this study was to investigate the effect of nisin and monensin on rumen fermentation of diets containing hay and barley (80:20%) in artificial rumen (Rusitec system). The Rusitec system consisted of four fermentation vessels (V1, V2, V3, V4): V1 was without additives (control), V2 received daily 2 mg of nisin, V3 involved 5 mg of monensin and V4 combination of 2 mg of nisin with 5 mg of monensin. After an adaptation period (7 days), the fermentation parameters were determined for six consecutive days. Compared to control diet, the addition of nisin resulted in an increase (P < 0.05) of hemicellulose degradation, acetate, propionate (mmol.day-1) production and energetic efficiency of VFA (E), decrease of butyrate production. Nisin had no effect on dry matter (DM), organic matter (OM), cellulose and detergent fiber degradation, production of total gas, methane and efficiency of microbial synthesis. The addition of monensin resulted in an decrease of DM, OM (P < 0.05), cellulose, hemicellulose, detergent fiber degradation (P < 0.001), total gas, methane and ammonia nitrogen (NH3-N) production. Monensin also significantly decreased acetate, butyrate, L-lactate (mmol.day-1) production and it increased propionate production (P > 0.001) and efficiency of microbial synthesis. The combined effect of nisin and monensin in V4 was similar to the effect of monensin in V3 compared to control. Then, the effect of additive monensin was dominant over nisin. In conclusion, our results indicate that nisin was less effective than monensin on some fermentation parameters (important for the improvement of the efficiency of utilization of the diet by ruminants) in artificial rumen.