The effect of adding plant extracts to ruminal fluid on the formation of conjugated linoleic acid (CLA) isomers and on fermentation parameters was evaluated. Initially, fifteen plant extracts at two levels (1 and 2 mg · ml^–1 buffered rumen fluid; BRF) were incubated with BRF and substrate (hay and concentrate, 50:50) at 39°C for 24 h, in a completely randomized block design. In the second experiment, four promising plant extracts affecting the formation of CLA and vaccenic acid (VA) were tested for their influence on basal parameters of rumen fermentation. The first study revealed that extracts of Azadirachta indica, Allium sativum, Cuminum cyminum at the lower level (1 mg · ml^–1) and extracts of Terminalia chebula at the higher level (2 mg · ml^–1) enhanced CLA isomer formation by 45.56%, 41.54%, 51.09%, and 15.54%, respectively, and the VA concentration by 10.97%, 10.82%, 14.93%, and 29.61%, respectively, in ruminal fluid when compared with the control (p < 0.05). The second experiment documented that the selected plant extracts did not impair nutrient fermentation. Nonetheless, addition of C. cyminum extract to ruminal fluid increased the partitioning factor (p = 0.054) and the digestibility of nutrients (p < 0.05). Furthermore, the polyunsaturated fatty acid concentration was higher when ruminal fluid was incubated with the C. cyminum extract (p = 0.049). In conclusion, extracts of Azadirachta indica, Allium sativum, Cuminum cyminum and Terminalia chebula modulate ruminal biohydrogenation and increase the concentrations of CLA isomers, their precursors, and VA without negatively affecting other rumen parameters. Cuminum cyminum did, however, improve the pattern of nutrient fermentation and can be considered a valuable supplement in ruminant nutrition to enhance the healthiness of ruminant meat and milk.