Rosemary extract (RE) is characterized as an antioxidant, and it has the potential to reduce methane emission and change microbial fermentation. Hence, to the extent of the evaluation of RE in ruminant nutrition, the in vitro fermentation technique was used to investigate the effects of RE on the fermentation characteristics of a total mixed ration (TMR) fed to dairy cows. Different doses of RE were added to the TMR to obtain different concentrations of antioxidants, including 0 (CON), 0.05 (LRE), and 0.10 g/kg (HRE). A total of 500 mg ground TMR was incubated in buffer solution and rumen fluid for 48 h at 39 °C. Nutrient degradability, gas production parameters, gas composition, fermentation parameters, and microbial composition were analyzed. The results showed that nutrient degradability and total volatile fatty acid concentration were not affected by the treatments. Furthermore, total methane production and proportion were depressed in a dose-dependent way. The RE increased the propionate concentration and proportion linearly and decreased the acetate concentration and proportion linearly. Finally, microbial diversity analysis showed that the richness and evenness indexes were unchanged by different treatments, while Prevotella_1 was decreased and Prevotella_7 was increased with RE supplementation. In conclusion, RE is an effective inhibitor of methane emission of microbial fermentation and changed the profile of volatile fatty acids with no disadvantageous effects on diet utilization.