This study was conducted to evaluate potential of Moringa oleifera seed oil (MOSO) to modulate rumen microflora to mitigate methane (CH4) production in different total mixed rations (TMRs). Three TMRs with different roughage (R) to concentrate (C) ratio were used as substrates (R70:C30, R50:C50 and R30:C70) for in vitro fermentation study using batch culture technique. Results revealed that supplementation of MOSO in different rations with variable roughage to concentrates ratio altered (P < 0.05) CH4 production and fermentation parameters. M. oleifera seed oil at 3 and 4% increased (P < 0.01) microbial protein (MCP) and propionate concentration for all rations but decreased acetate concentration in R70:C30 TMR. Two levels of MOSO (3 and 4%) quadratically increased (P = 0.001) MCP and decreased acetate in R30:C70 while all levels increased propionate. For R30:C70 and R50:C50 rations, MOSO linearly and quadratically decreased (P < 0.001) protozoa and methanogen counts; however, protozoa, methanogens and bacteria were significantly increased in R70:C30 ration without any change in fungal counts. Supplementation of higher level of MOSO (4%) in high roughage ration (R70:C30) showed negative effects on diversity (Shannon index) and evenness of bacterial species as compared to control and lower oil level. Moreover, it also decreased Firmicutes to Bacteroidetes ratio in high roughage rations more obviously at lower levels. Moringa oil also stimulated Prevotella in both high and low roughages diets that indicates its potential to mediate rumen acidosis. Conclusively, MOSO enhanced fermentation kinetics and decreased CH4 production through effective modulation of rumen microbiome.