This study aimed to investigate the effect of supplementing Isochrysis galbana (I. galbana) at levels of 0 (control), 1, 2, 3, 4, and 5 (g/100 g DM) of the diet on the gas production kinetics, methane production, rumen fermentation parameters, and relative microbial population in vitro. Supplementation of I. galbana at high level (5g/100 g DM) caused a significant decrease in total gas production (p < 0.05). High supplementation rates (4 and 5g/100 g DM) decreased CH4 production relative to the control by 18.4% and 23.2%, respectively. Although rumen ammonia nitrogen (N-NH3) and total volatile fatty acids (VFA) concentrations were affected by dietary treatments, but the VFA profile did not changed. The relative proportion of protozoa and methanogenic archaea as well as Anaerovibrio lipolytica, Prevotella spp., Ruminococcus flavefaciens, and Fibrobacter succinogenes were decreased significantly as a result of microalgae supplementation. However, the relative abundance of Ruminococcus albus, Butyrivibrio fibrisolvens and Selenomonas ruminantium were significantly increased (p < 0.05), related to the control group. As well, the pH was not affected by dietary treatments. It was concluded that I. galbana reduced in vitro CH4 production and methanogenic archaea that its worth to be investigated further in in vivo studies.