The experiment was conducted to determine effects of urea-lime-treated rice straw and urea levels in concentrate on rumen fermentation, apparent nutrient digestibility, and cellulolytic bacteria population of 4-year-old, rumen-fistulated swamp buffaloes. All animals were randomly assigned according to a 2 × 2 factorial arrangement in a 4 × 4 Latin square design to receive four dietary treatments: factor A, two sources of roughage (rice straw and 2%urea + 2%lime-treated rice straw); factor B, two levels of urea in concentrate mixture (0% and 4%). Roughages were given ad libitum together with 0.3% BW of concentrate. It was found that voluntary feed intake, the digestibility of DM, OM, CP, NDF, acetate, and propionate concentration were significantly increased (P < 0.05) by treated rice straw, while NH(3)-N, BUN, and propionic acid concentration were increased by both factors of treated rice straw and 4% urea in concentrate. The real-time PCR quantification of Fibrobacter succinogenes and Ruminococcus albus population, and anaerobic fungi were greater (P < 0.05), but the population of Ruminococcus flavefaciens, protozoa, and methanogenic bacteria were reduced (P > 0.05) as influenced by treated rice straw and urea level. In conclusion, the combined use of urea-lime-treated rice straw and fed with concentrate (4% urea) could improve rumen ecology, rumen fermentation efficiency, and nutrient digestibility in swamp buffaloes.