Trans isomers of fatty acids exhibit different health properties. Among them, trans-10,cis-12 conjugated linoleic acid has negative effects on milk fat production and can affect human health. A shift from the trans-11 to the trans-10 pathway of biohydrogenation (BH) can occur in the rumen of dairy cows receiving high-concentrate diets, especially when the diet is supplemented with highly unsaturated fat sources. The differences of BH patterns between linoleic acid (LeA) and linolenic acid (LnA) in such ruminal conditions remain unknown; thus, the aim of this work was to investigate in vitro the effects of starch and sunflower oil in the diet of the donor cows and starch level in the incubates on the BH patterns and efficiencies of LeA and LnA. The design was a 4 × 4 Latin square design with 4 cows, 4 periods, and 4 diets with combinations of 21 or 34% starch and 0 or 5% sunflower oil. The rumen content of each cow during each period was incubated with 4 substrates, combining 2 starch levels and either LeA or LnA addition. Capillary electrophoresis single-strand conformation polymorphism of incubates showed that dietary starch decreased the diversity of the bacterial community and the high-starch plus oil diet modified its structure. High-starch diets poorly affected isomerization and first reduction of LeA and LnA, but decreased the efficiencies of trans-11,cis-15-C18:2 and trans C18:1 reduction. Dietary sunflower oil increased the efficiency of LeA isomerization but decreased the efficiency of trans C18:1 reduction. An interaction between dietary starch and dietary oil resulted in the highest trans-10 isomers production in incubates when the donor cow received the high-starch plus oil diet. The partition between trans-10 and trans-11 isomers was also affected by an interaction between starch level and the fatty acid added to the incubates, showing that the trans-10 shift only occurred with LeA, whereas LnA was mainly hydrogenated via the more usual trans-11 pathway, whatever the starch level in the substrate, although the bacterial communities were not different between LeA and LnA incubates. In LeA incubates, trans-10 isomer production was significantly related to the structure of the bacterial community.