Fermented total mixed ration (TMR) is a novel feed for ruminants in South Korea. The purpose of this study was to evaluate the effects of lactic acid bacteria (LAB) on the quality of TMR and in vitro ruminal fermentation. Strains of three LAB spp. (Lactobacillus plantarum, L. brevis, L. mucosae) were used in fermentation of TMR. Inoculations with the three LAB spp. lowered pH and increased concentrations of lactic acid, acetic acid, and total organic acid compared to non-LAB inoculated control (only addition of an equivalent amount of water) (p<0.05). Bacterial composition indicated that aerobic bacteria and LAB were higher. However, E. coli were lower in the fermented TMR than those in the control treatment (p<0.05). Among the treatments, L. brevis treatment had the highest concentration of total organic acid without fungus detection. Gas production, pH, and ammonia-nitrogen during ruminal in vitro incubation did not differ throughout incubation. However, ruminal total VFA concentration was higher (p<0.05) in the LAB spp. treatments than the control treatment at 48 hours. Overall, the use of L. brevis as an inoculant for fermentation of high moisture. TMR could inhibit fungi growth and promote lactic fermentation, and enhance digestion in the rumen.