This study estimated the effects of oil sources on fermentation characteristics, greenhouse gas, microbial diversity, and biohydrogenation of fatty acids in the rumen. In vitro ruminal incubation was performed with 7 mg of oil source, 15 mL rumen buffer, and 150 mg of synthetic diet at 39 °C for 0, 3, 6, 12, and 24 h. Oil sources consisted of corn oil (CO; linoleic acid (C18:2n-6)), linseed oil (LSO; linolenic acid (C18:3n-3)), or Ca-salts (protected C18:2n-6). The ruminal gas was collected for CH4 and CO2 analysis. Incubated rumen buffer was sub-sampled for the analysis of microbial quantification, fermentation characteristics, and fatty acid profiles. The results showed that Ca-salt increased acetate (p = 0.013), while CO increased propionate (p = 0.007). Fibrobacter succinogenes, Ruminococcus flavefaciens, and R. albus increased (p < 0.05) with Ca-salt after 12 h of incubation, while Streptococcus bovis increased (p < 0.05) by LSO. The CO and Ca-salt resulted in the highest C18:2n-6 (p = 0.002), while LSO resulted in the highest C18:3n-3 (p = 0.001). The Ca-salt had the lowest C18:0 (p = 0.002), but the highest C18:1cis-9 (p = 0.004). In conclusion, Ca-salt supplementation resisted biohydrogenation to some extent, decreased methanogenic archaea and protozoa, and exerted less toxic effects on fibrolytic bacteria.