The considerable contribution of ruminant livestock to methane emissions has become a major global concern in recent years. Although dietary approaches for reducing ruminant methane emissions have been explored, the sustainable potential of probiotics to influence rumen function and lower methane production has increasingly attracted research attention. While previous studies have focused on single or dual-strain probiotics, this study is among the first to evaluate the synergistic effects of quadric-strain formulations. Hence, this study aimed to evaluate the impact of multi-strain probiotic blends, each at two distinct concentrations on rumen fermentation, nutrient degradability, and methane emission in sheep using an in vitro gas production technique following a completely randomized design. The basal diet with no probiotic supplements served as a control, while the supplemented bacterial combinations were Bacillus licheniformis, Lactobacillus acidophilus, L. bulgaricus, and Bifidobacterium bifidum (ABLB; at a ratio of 1:1:1:1) at levels of 2 × 109 (ABLB2) and 4 × 109 (ABLB4) CFU/g of feed, and Lactobacillus casei, Lactobacillus plantarum, Bacillus subtilis plus Bifidobacterium bifidum (CPSB; at a ratio of 1:1:1:1) at levels of 2 × 109 (CPSB2) and 4 × 109 (CPSB4) CFU/g of feed. Probiotic supplementation significantly improved in vitro dry matter and fiber degradability (IVDMD and IVCFD), with the most effective results observed in ABLB treatments. These blends also reduced methane production and ammonia-N concentrations, while increasing total volatile fatty acids (TVFA), indicating more efficient fermentation. Protozoa counts were notably lower in treated groups, supporting the role of probiotics in mitigating methane via microbial modulation (P < 0.01). Probiotic supplementation did not affect the values of pH (P > 0.05). Predictive values for metabolizable energy (ME), net energy for lactation (NEL), and organic matter digestibility (OMD) were improved across treatments. These findings highlight the potential of targeted probiotic formulations to enhance rumen efficiency and reduce