The livestock industry significantly contributes to greenhouse gas emissions, with ruminant animals, including cows, sheep, and goats, being responsible for a substantial share of these emissions due to methane production. Reducing methane emissions from ruminants is crucial for mitigating the environmental impact of livestock production. Additionally, there has been a growing interest in improving the nutritional quality of ruminant products through modifying their profile of fatty acids. The current study aimed to investigate the potential of sage (SAG), pine (PIN), and clove (CLO) essential oils as natural additives for modulating in vitro ruminal fermentation characteristics and biohydrogenation of polyunsaturated fatty acids (PUFA). Within the current experiment, three dose levels (300, 600, and 900 mg/L) of essential oils were evaluated using rumen inoculum from three mature Dalagh ewes (58 ± 2.84 kg body weight). The results revealed that the essential oils had a significant impact on gas production, methane and carbon dioxide production, ruminal fermentation parameters, and ruminal biohydrogenation of dietary PUFAs. The essential oil treatments resulted in reduced gas production compared with the control group. Methane production was significantly reduced by all doses of the essential oils, with the highest dose of CLO resulting in the lowest methane production. In addition, the essential oils affected ruminal fermentation parameters, including pH, ammonia concentration, and production of total volatile fatty acids. Promising modifications in ruminal biohydrogenation of PUFAs and the profile of fatty acids were also observed in the current study. These findings suggest that SAG, Pin, and CLO hold promise in mitigating methane emissions and improve the nutritional value of ruminant products. Further investigation is required to evaluate their effectiveness in practical feeding strategies for livestock.