Abstract: The aim of this study was to evaluate the relationship of rumen anaerobic fungi and methanogenic archaea on ruminal fermentation in swamp buffalo. Four rumen fistulated swamp buffaloes with average 369 kg of body weight were used. All animals were randomly assigned according to a 2x2 factorial arrangement in a 4x4 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 3 g kg^-1 BW of concentrate. It was found that voluntary feed intake, the digestibility of DM, CP, NDF, acetate and propionate concentration were significantly increased (p<0.05) by treated rice straw while NH₃-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 F. succinogenes and R. albus population and anaerobic fungi were greater (p<0.05) but the population of R. flavefaciens, protozoa and methanogenic bacteria were reduced (p>0.05) as influenced by treated rice straw and urea level. Animal consumed treated rice straw was shown in more diverts in phylogenetic relationship. No change in rumen methanogenic bacteria diversity and relative change with fungi population. 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 increase anaerobic fungi. The results from this study suggest that feeding with urea-lime treated rice straw more rumen fermentation efficiency though shift fungi and methanogenic population.