Plant bioactive compounds have been chosen as alternatives to antibiotics to promote animal productivity. Biochanin A is a type naturally occurring bioactive compound. It is O-methylated isoflavone and found in red clover, alfalfa sprouts, and other legumes. The aim of this study was to determine the effect of biochanin A on rumen microbial fermentation and composition. The experimental design consisted of a control (no additive) and biochanin A (0.03 mg/mL) and each treatment was triplicated. Anaerobic medium was prepared with sterile rumen fluid and McDougall buffer in tubes. The tubes containing anaerobic medium were inoculated with rumen mixed microbes, and cultured at 39°C for 24 h as the first generation. Then, the culture from the first generation was transferred by inoculation to new anaerobic tubes with the medium incubated as the second generation. The transferring was continued until the fourth generation. The fermentation fluid was sampled for the detection of VFA, ammonia and amino acids at 0, 12, 16, 24 h during culturing. The gas production in the tube was detected by pressure sensor. Different doses of biochanin A were mixed with urea to evaluate its inhibition of urease activity and calculate half-maximal inhibitory concentration (IC50). The bacterial composition was carried out by 16S rRNA gene sequencing. The significant differences between 2 treatments were analyzed by ANOVA in SAS. Results showed that biochanin A increase microbial gas production by 2.5% (P < 0.05), but no effect on VFA production. Microbial urease activity was inhibited (P < 0.05) by biochanin A with the IC50 of 319.8 μmol/L. Biochanin A also inhibited (P < 0.05) the degradation rate of Val, Lys, Met, Leu and total amino acids by 55.84, 37.03, 12.63, 11.74, and 18.00%, respectively. Inhibition of urease activity and amino acid decomposition by biochanin A resulted in a reduction (P < 0.05) of ammonia by 28%. The 16S rRNA gene sequencing showed that biochanin A reduced (P < 0.05) the abundance of proteolytic bacteria Prevotella and Streptococcus. Therefore, biochanin A reduced the production of ammonia by inhibiting proteolytic bacteria and its decomposition of urea and amino acids activity.