Abstract: Four rumen-fistulated, male swamp buffalo were randomly assigned according to a 4x4 Latin square design to evaluate the effect of the urea-treated rice straw to concentrate ratio (R:C) on rumen fermentation, nutrient digestibilities, microbial protein synthesis and cellulolytic bacterial population. Animals were fed R:C of 100:0, 75:25, 50:50 and 25:75, respectively. Results showed that digestibility of nutrients were significantly affected by R:C especially those of OM and fiber. However, digestibility of CP, ruminal NH₃-N and plasma urea N were similar among treatments (p>0.05) whereas ruminal pH was decreased significantly (p<0.01) when concentrate ratio was increased. Total VFA concentrations and C3 were significantly different among treatments and were greatest at 50:50 of R:C supplementation (p<0.01). Total viable bacteria, proteolytic bacteria and bacteria cell count were not altered among treatments (p>0.05) whereas amylolytic bacteria, cellulolytic bacteria and fungal zoospore were significantly different (p<0.01), responding to a change in proportion of R:C. Moreover, using of real-time PCR technique provided that feeding of a 100% roughage remarkably increased these three cellulolytic bacteria numbers up to 3.54x10⁹ copies mL^-1 for F. succinogenes, 7.38x10⁷ copies mL^-1 for R. Flavefaciens and 5.80x10⁶ copies mL^-1 for R. albus in rumen digesta, respectively. It is most notable that F. succinogenes were the highest in population in the rumen of swamp buffalo. In addition, efficiency of rumen microbial N synthesis were enriched by R:C supplementation, especially at the ratio of 50:50 (p<0.05). Based on this study, it could be concluded that supplementation of R:C at 50:50 improved digestibilities of nutrients, ruminal ecology and microbial protein synthesis efficiency.