Tea seed oil (TSO) was investigated for its effects on rumen fermentation and in vitro parameters of bacterial communities in water buffalo diets containing Siraitia grosvenorii and soybean residues. TSO was added at rates of 0% (control group (CT)), 0.5% (T1), 1% (T2), and 2% (T3) of the in vitro fermentation substrate weight (dry matter (DM) basis). T2 and T3 had significantly lower acetate and total volatile fatty acid contents but a significantly higher microbial crude protein content than CT. The lowest NH3-N content was observed in T1 and T2. Treatment significantly increased DM digestibility, with the highest percentage observed in T2. T2 showed significantly higher crude protein digestibility than CT. TSO supplementation significantly increased the C18:2n6c, C18:2 trans-10, cis-12, and C20:4n6 concentrations compared to those in CT. The total number of bacteria was significantly lower in T2 than in CT. TSO supplementation decreased the total bacteria, fungi, and methanogen populations but increased rumen microorganism diversity and richness. In conclusion, TSO can regulate the number and flora of rumen microorganisms through antimicrobial activity, thereby affecting rumen fermentation patterns, reducing methane production, and improving nutrient digestibility, and an optimal supplementation rate appears to be achieved with 1% TSO (DM basis).