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The aim of this study was to evaluate the effect of Natuzyme enzyme mixture (0, 3, and 6 g/kg dry matter [DM]) on microbial digestion as well as fermentation of wheat straw and alfalfa hay in Arabian horse. Four female Arabian horses (3–4 years, average body weight 400 kg) were fed with diet containing 35% commercial concentrate +65% forage (wheat straw and alfalfa hay) for 2 weeks. The fermentation and gas production of wheat straw and alfalfa hay with different levels of Natuzyme enzyme were determined using standard in vitro gas production protocol (6 replicates). The DM and neutral detergent fiber (NDF) digestibility were determined using specific media (four replicates per time). Addition of enzyme had no effect on gas production rate of alfalfa hay (P > .05), but the potential of gas production was increased with alfalfa hay at 3 and 6 g/kg DM of enzyme compared with control. The addition of enzyme to alfalfa did not affect partitioning factor, microbial biomass, microbial biomass efficiency, and cell wall degradability (P > .05). DM digestibility of alfalfa hay after 24, 48, and 72 hours of incubation with mixed cecum contents was not affected by enzyme (P > .05), but NDF digestibility at 24, 48, and 72 hours was increased (P < .05). The enzyme supplementation decreased microbial biomass efficiency and increased potential of gas production, DM, and NDF digestibility at 24 hours for straw. The highest value was obtained for straw treated with 3 and 6 g/kg DM of enzyme. Gas production parameters, DM, and NDF digestibility of straw at 48 and 72 hours of incubation were not influenced by enzyme addition. In conclusion, addition of Natuzyme enzyme mixture caused the proper fermentation, gas production, and digestibility of alfalfa hay and straw without affecting the DM digestibility by cecal microorganisms, thereby suggesting the additive role of this enzyme in Arabian horse nutrition.Enzyme had improved potential of gas production of alfalfa at 3 and 6 g/kg dry matter.The neutral detergent fiber digestibility at 24, 48, and 72 hours was increased with enzyme addition.Enzyme addition improved microbial biomass efficiency and gas production for straw.Dry matter and neutral detergent fiber digestibility at 24 hours with enzyme supplementation in straw.