The aim of this study was to investigate the effects of sugarcane neutral detergent fiber digestibility (NDFD), conservation method, and concentrate level on the ruminal microbial population of steers. Eight ruminal-cannulated Nellore steers were distributed in two contemporary 4 × 4 Latin Square design with a 2 × 2 factorial arrangement of treatments. Experiment 1: diets were formulated with 60% of concentrate level, and two sugarcane genotypes (high or low NDFD) either freshly cut or as silage. Experiment 2: diets were formulated with two levels of concentrate (60 or 80%), and two sugarcane genotypes (high or low NDFD) offered as freshly cut. Each experimental period lasted for 14 d, with the last 4 d used for ruminal fluid collection. Three cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens), two amylolytic (Streptococcus bovis, Ruminobacter amylophilus), and a lactate fermenting microorganism (Megasphaera elsdenii) were quantified by qPCR. Experiment 1: diets with fresh sugarcane increased the population of S. bovis, and M. elsdenii. Sugarcane with high NDFD increased F. succinogenes population only when sugarcane was offered as freshly cut. Experiment 2: increasing concentrate in the diet decreased S. bovis population, and increased R. amylophilus. Sugarcane with high NDFD increased the population of cellulolytic bacteria only at the 60% concentrate diet. Providing sugarcane with high NDFD favored the growth of fibrolytic bacteria, and this effect were dependent on the conservation method and on diet concentrate level. In addition, sucrose appears to have great effect on the composition of ruminal microflora, especially S. bovis.