Polyunsaturated fatty acids (PUFAs) constitute a significant lipid class with essential nutritional and health benefits for both animal and human health; however, their effect and interaction with the gut microbiota ecosystem are still unclear. Therefore, the present study aims to investigate the effect of fish oil (FO) on ruminal fermentation and bacterial abundance under high- and low-forage diets. Thirty-six ruminal fluid samples were allocated into two experiments. The first was on high-forage diet and included three groups: the control (basal diet with 70% forage and 30% concentrate), group 2 (basal diet + 5 mL/L FO), and group 3 (basal diet + 10 mL/L). The second experiment was on low-forage diet: the control (basal diet with 30% forage and 70% concentrate), group 2 (basal diet + 5 mL/L FO), and group 3 (basal diet + 10 mL/L). The results showed that although FO supplementation did not affect the pH level among different diets, it significantly decreased methane under a high-forage diet. In addition, regarding the fatty acids profile, FO supplementation in high-forage diet significantly decreased fatty acids in both; however, under a low-forage diet, FO groups showed significantly higher fatty acid content than the control. However, FO supplementation increased the abundance of Anaerovibirio, Selenomonas, pseudobutyrivibrio, and butyrivibrio through a high-forage diet. In contrast, the abundance of Prevotella, Rikenellaceae RC9 gut group, and Saccharofermentans was depressed with FO supplementation. Whereas under low-forage diet, FO supplementation increased Ruminobacter, Anaerovibirio, Megasphaera, Pseudobutyrivibrio, Streptococcus, Butyrivibrio, unclassified_lachnospiraceae; it also decreased Prevotella and Rikenellaceae RC9 abundance similar to the high-forage diet. Based on the KEGG pathway results, FO supplementation significantly downregulated genes mainly related to folding, sorting and degradation, environmental adaptation, cell motility, transcription, membrane transport, and signal transduction. The results revealed that FO has a depressing effect on ruminal fermentation and some bacterial population; however, this negative effect can be minimized in high-concentrate diets.