Modifying ruminal biohydrogenation (BH) is a widely investigated approach to improve the fatty acid (FA) profile of ruminant-derived products. This study evaluated the effects of thyme (Thymus vulgaris; TEO), peppermint (Mentha piperita; PEO), and lavender (Lavandula angustifolia; LEO) essential oils (EOs) on in vitro ruminal short-chain fatty acid (SCFA) profiles and BH of polyunsaturated fatty acids (PUFAs). A basal substrate consisting of alfalfa hay and a concentrate mixture (60:40) supplemented with 2% fish oil (FO) was used as a source of long-chain PUFAs. The treatments included a control (FO-supplemented TMR without EO) and three EO groups, each added at 1.5 µl/ml of buffered rumen fluid. All EOs significantly inhibited the final step of BH, as indicated by a marked reduction in stearic acid (C18:0) concentration (P < 0.01). TEO supplementation resulted in the highest concentrations of c9, t11-CLA, and linoleic acid, suggesting inhibition of the final BH step and greater availability of vaccenic acid for endogenous CLA synthesis. Conversely, PEO resulted in the highest concentrations of eicosapentaenoic acid (EPA, C20:5 n-3) and docosahexaenoic acid (DHA, C22:6 n-3) (P < 0.01), together with the lowest concentration of palmitic acid (C16:0), indicating greater protection of fish oil-derived PUFAs. TEO also increased the acetate-to-propionate ratio (P < 0.01). Our findings demonstrate that the effects of the essential oils on ruminal BH differed between plant species. TEO promoted CLA accumulation, whereas PEO more effectively preserved EPA and DHA and reduced palmitic acid concentration.