Enterococcus faecalis is one of the beneficial microorganisms which produces enterolysin and converts fumarate to succinate. Hence, this study was conducted to determine the effect of adding E. faecalis on in vitro ruminal fermentation, methane concentration, microbial diversity and population. Ruminal samples were collected from ruminally cannulated Holstein Friesian cattle, and a substrate consisting of rice straw and concentrate mixture at 40:60 DM was offered at 1g DM/100mL buffered ruminal fluid. Fresh culture of E. faecalis (7.5 × 10⁸ cfu/mL) at different inclusion rates were investigated using in vitro ruminal fermentation. The treatments tested were: non addition (control), 0.1% (T1), 0.5% (T2), and 1.0% (T3) of E. faecalis. All treatments were conducted in triplicates and analyzed by ANOVA for randomized complete block design. Duncan’s Multiple Range Test (DMRT) and Orthogonal Polynomial Contrast were used to identify differences among treatments and control. All analyses were carried out using SAS version 9.1 (SAS, 2002). Total gas production and ammonia nitrogen concentration of cultures were directly proportional to incubation times, while culture pH was inversely proportional to incubation times. Addition of E. faecalis lowered (P < 0.05) the total gas production after 24 h but the opposite was observed after 48 h of incubation having the highest (P < 0.05) in T3 followed by T4, T2 and control with 93.80, 91.00, 88.33 and 82.20 ml, respectively. Higher concentrations of acetate were detected in E. faecalis treatments than control at 12 and 48 h but the opposite was observed at 24 h, while propionate was highest (P < 0.05) in T3 after 48 h with 12.59 mM. Butyrate concentration was higher (P < 0.05) in E. faecalis treatments than control after 12 and 24 h of incubations and the highest (P < 0.05) concentrations were detected in T2 at 24 h and T3 at 48 h with 14.61 and 14.88 mM, respectively. Higher (P < 0.05) concentrations in E. faecalis treatments than control were also observed in total volatile fatty acid (VFA) concentration after 12 and 48 h. Addition of E. faecalis enhanced in vitro ruminal fermentation by increasing the butyrate and total VFA concentrations in dose dependent manner.