The effect of crude glycerin (CG) - 80% of glycerol - inclusion as a substitute to corn grain in supplements on rumen fluid protozoa numbers, and relative proportion of cellulolytic bacteria and methanogenic archaea of rumen-cannulated Nellore steers (n = 10; BW = 490 ± 47 kg) grazing tropical grass, during the rainy season was studied. Treatments were constituted by 5 levels of CG in the supplement: 0, 7, 14, 21 and 28% DM of CG. Animals were supplemented daily at 1000 h in a proportion of 300 g/100 kg of BW. Supplement consisted of corn grain, soybean meal, urea, gluten meal and mineral mix. Bacteria and protozoa samples were collected on the d 11 of each experimental period, 3 h after supplementation, in the solid and liquid extracts in different parts of the rumen. Real-time PCR was used to quantify microbial population. Methanogens and cellulolytic bacteria were expressed as a proportion (%) of total rumen bacterial 16S rDNA. Ciliate protozoa species were identified and quantified (#/mL) in a Sedgewick-Rafter chamber. Data of protozoa were log10-transformed and analyzed in a replicated Latin square design using the MIXED procedure of SAS. Bacterial proportions were analyzed using the software R, with data compared between treatments (with or without 28% DM of CG in the supplement) using the Wilcoxon test, the effects of treatments were considered significant at P < 0.05. The inclusion of crude glycerin in supplement did not affect (P > 0.05) rumen protozoa number (6.8, 5.6, 5.2, 2.4, 2.0, and 3.6 for the genera Entodinium, Dasytricha, Isotricha, Eremoplastron, Diploplastron, and Polyplastron). The inclusion of CG decreased the relative proportion of Ruminococcus albus (P = 0.047) and Ruminococcus flavefaciens (P = 0.036), with mean values of 0.0702 and 0.0174; 0.0411 and 0.0073 respectively for 0% DM and 28% DM of CG in the supplements. However, had no effect on Fibrobacter succinogenes (P = 0.420), and methanogens (P = 0.150) with mean values of 0.0199 and 0.1791, respectively. Inclusion of CG at the level of 28% of DM in the supplement negatively affect the rumen cellulolytic bacteria R. albus and R. flavefaciens of Nellore steers grazing tropical grass.