With the increasing adoption of sugarcane silage in ruminant feed, it becomes essential to understand the effects of supplementation with different levels and sources of non-fiber carbohydrates (NFC) on ruminal microorganisms. The aim of this study was to characterize the population change of cellulolytic and amylolytic rumen bacteria, caused by the increase of concentrate, and by the use of different sources of NFC in diets with sugarcane silage. Six Nellore beef cattle, castrated, and cannulated in the rumen, were used in a 3 0215 3 Latin square design. The experimental diets were formulated with 2 levels of concentrate: 60% or 80%, and the roughage used was sugarcane silage. Within each level of concentrate inclusion, 3 different sources of NFC were used: steam flaked corn (SFC), pelleted citrus pulp (PCP), or ground corn (GC). SFC and PCP were included in the diet in partial replacement of 70% of GC. At d 14 of each period, samples of rumen contents were collected for DNA extraction and subsequent analysis of the relative quantification of rumen microorganisms by real time PCR. The increase of concentrate in the diet resulted in a decrease in population of Fibrobacter succinogenes (P < 0.01) and Streptoccocus bovis (P < 0.01), and in an increased of Ruminoccocus flavefaciens (P = 0.05) and Megasphaera elsdenii population (P = 0.04), without changing the Ruminoccocus albus population (P = 0.63). The partial replacement of GC by PCP resulted in an increase of S. bovis population (P = 0.01) and in a reduction of R. flavefaciens (P < 0.01), without changing F. succinogenes (P = 0.64). Moreover, the replacement of GC by SFC increased the population of M. elsdenii (P = 0.03) and reduced R. albus (P < 0.01). There was a significant Diet*NFC interaction only for M. elsdenii (P = 0.02), where SFC increase M. elsdenii population only at the 80% concentrate diet. In conclusion, substituting GC for PCP resulted in an increase only in population of S. bovis, while substituting GC for SFC resulted in a decrease in population of R. albus, and in an increase of M. elsdenii, in diets with sugarcane silage as the roughage source.