Effects of starch content (SC) and fermentability (SF), and culture pH on biohydrogenation (BH) of unsaturated fatty acids (FA) and NDF digestibility (NDFD) in batch culture were evaluated. Cultures (4 replicates/treatment/time point) were incubated at pH 5.8 or 6.2 for 0, 12, and 24 h and included alfalfa hay (55 or 70% of DM), and either dry ground corn (DC) or high moisture corn (HMC) as starch sources (30 or 45% of DM) for a SC of 22 or 33% DM. The alfalfa hay was treated with corn oil (2% of DM) to increase the total unsaturated FA content of cultures. Effects of SC, SF, culture pH, and their interactions were determined. BH extent for cis-9,cis-12 18:2 and NDFD increased over time across all treatments (both P < 0.001). For main effects of treatments, 33% SC compared with 22% SC and pH 5.8 compared with pH 6.2 reduced BH extent of cis-9,cis-12 18:2 (35 vs. 39% and 23 vs. 51%, respectively; both P < 0.001) and NDFD (13 vs. 16% and 8 vs. 21%, respectively; both P < 0.001). Compared with DC, HMC decreased NDFD (13 vs. 16%; P < 0.001). HMC increased BH extent of cis-9,cis-12 18:2 at pH 5.8, and decreased it at pH 6.2 compared with DC (interaction P < 0.001) with no overall effect of SF (P = 0.90). Contents of trans-10,cis-12 18:2 (CLA) and trans-10 18:1 increased over time for all treatments (both P < 0.001). Overall, 33% SC compared with 22% SC and pH 5.8 compared with pH 6.2 increased content of CLA by 20 and 67% respectively (both P < 0.001). Although SF did not affect CLA content overall (P = 0.13), it interacted with time (interaction P < 0.001); HMC increased CLA at 12 h, and decreased it at 24 h compared with DC. The 33% SC compared with 22% SC, HMC compared with DC, and pH 5.8 compared with pH 6.2, increased content of trans-10 18:1 by 17, 28, and 43% respectively (all P < 0.001). In conclusion, higher SC and lower culture pH decreased BH of cis-9,cis-12 18:2 and NDFD, and increased formation of CLA and trans-10 18:1. Compared with DC, HMC increased formation of trans-10 18:1, decreased NDFD, and interacted with culture pH and time for BH of cis-9, cis-12 18:2 and the formation of CLA.