Objective: This study aimed to determine the effects of different roughages in total mixed ration (TMR) inoculated with or without coculture of <i>Lactobacillus acidophilus</i> (<i>L. acidophilus</i>) and <i>Bacillus subtilis</i> (<i>B. subtilis</i>) on <i>in vitro</i> rumen fermentation and microbial population.Methods: Three TMRs formulations composed of different forages were used and each TMR was grouped into two treatments: non-fermented TMR and fermented TMR (F-TMR) (inoculated with coculture of <i>L. acidophilus</i> and <i>B. subtilis</i>). After fermentation, the fermentation, chemical and microbial profile of the TMRs were determined. The treatments were used for <i>in vitro</i> rumen fermentation to determine total gas production, pH, ammonianitrogen (NH<sub>3</sub>-N), and volatile fatty acids (VFA). Microbial populations were determined by quantitative real-time polymerase chain reaction (PCR). All data were analyzed as a 3×2 factorial arrangement design using the MIXED procedure of Statistical Analysis Systems.Results: Changes in the fermentation (pH, lactate, acetate, propionate, and NH<sub>3</sub>-N) and chemical composition (moisture, crude protein, crude fiber, and ash) were observed. For <i>in vitro</i> rumen fermentation, lower rumen pH, higher acetate, propionate, and total VFA content were observed in the F-TMR group after 24 h incubation (p<0.05). F-TMR group had higher acetate concentration compared with the non-fermented group. Total VFA was highest (p<0.05) in F-TMR containing combined forage of domestic and imported source (F-CF) and F-TMR containing Italian ryegrass silage and corn silage (F-IRS-CS) than that of TMR diet containing oat, timothy, and alfalfa hay. The microbial population was not affected by the different TMR diets.Conclusion: The use of Italian ryegrass silage and corn silage, as well as the inoculation of coculture of <i>L. acidophilus</i> and <i>B. subtilis</i>, in the TMR caused changes in the pH, lactate and acetate concentrations, and chemical composition of experimental diets. In addition, F-TMR composed with Italian ryegrass silage and corn silage altered ruminal pH and VFA concentrations during <i>in vitro</i> rumen fermentation experiment.