The influence of coconut oil on microbial activity was investigated in vitro using a rumen simulation technique (Rusitec). Diets containing either 40 (L-diet) or 60% (H-diet) of concentrate and 60 or 40% hay, respectively, were supplemented with coconut oil at the rate of 5% DM (experimental cultures) or given alone (control cultures). Coconut oil significantly reduced ciliate numbers and methane concentration independently of the diet (P < 0.05). Bacteria numbers increased significantly (L-diet) or remained unaffected (H-diet). And, conversely, the ammonia concentration remained unchanged (L-diet) or decreased (H-diet). Coconut oil had no effect on the molar proportions of acetate, propionate, or butyrate.