This study investigated the effects of different probiotic mixes on fermentation parameters, methane emissions, and nutrient degradability of the diet in vitro. The examined bacterial probiotic mixes were 1- Lactobacillus acidophillus + Lactobacillus bulgaricus (AB), 2- Lactobacillus cassia + Lactobacillus plantrum (CP), 3- Bacillus lichnoformas + Bacillus subtillus (LS), 4- Lactobacillus acidophillus + Lactobacillus bulgaricus + Bacillus lichnoformas (ABL), 5- Lactobacillus cassia + Lactobacillus plantrum+ Bacillus subtillus (CPS),6- Lactobacillus acidophillus + Lactobacillus bulgaricus + Bacillus lichnoformas + Bifidobuctrium bifidum (ABLB) and 7- Lactobacillus cassia + Lactobacillus plantrum+ Bacillus subtillus + Bifidobuctrium bifidum (CPSB). The probiotic mixes were used at levels 0 (control), 2, and 4×109 cfu /g feed. Significant effects were observed in gas production across all incubation times, with the CPS mix exhibiting the highest production after 48 hours. Methane emissions significantly decreased with all mixes of probiotics, with the CP mix demonstrating the most substantial reduction. The degradability of dry matter and crude fiber was significantly influenced by supplementation levels, peaking at 2 and 4 × 109 cfu/g feed. Total volatile fatty acid (TVFA) production was significantly affected, with ABLB and CPSB mixes producing the highest TVFA. Furthermore, significant effects of supplementation levels were noted on ammonia-N and TVFA production. Additionally, pH value was significantly affected by the mixes of probiotics and supplementation levels. In conclusion, the probiotic combinations enhanced the rumen fermentation and degradability. besides, it reduced the methane emission. So, it is able to be used in an applicable in vivo study.