Distribution patterns of adherent microorganisms lining the digestive tract and gut wall, suggest an intimate interaction between the colonizing fungi and their hosts. Small numbers of bacteria preferentially live in the rumen fluid; However, the most unique population of bacteria form adherent biofilms on the surface of the stratified squamous epithelium of the rumen wall. This latter community contain many facultative anaerobic organisms that consume the oxygen which diffuses from the rumen wall tissue and thus protect the fastidious anaerobes within the rumen. Since the proportion of blood flowing to the ventral rumen is double that of flowing to the dorsal rumen, it is likely that along with the greater flow, there would be an equivalent increase in the population of rumen facultative microorganisms. This study was undertaken to isolate and identify prevascular rumen fungi and to evaluate their enzymatic activity profiles. Six sheeps were used in this study. Three sheeps received a concentration feed, and the other three received highly cellulosic feed. After two weeks, the animals were sacrificed and tissue blocks were removed from prevascular reticulo-rumen locations under sterile condition. Also, tissue samples were obtained from locations without vasculature as control. All of the samples were incubated on solid cultures. This is the first study to report isolation of four new facultative anaerobic fungi identified as Aspergillus terreus , A. flavus , A. penicilloides and Trichoderma longibrachiatum from the rumen wall fed cellulosic diet and one new fungus identified as Absidia corymbifera from the rumen wall of sheep fed concentrate. The fungus A.corymbifera is reported for the first time for IRAN. All of the isolated fungi exhibited cellolulytic, amylolytic and proteolytic activities. Except A.corymbifera , other isolated fungi were found to hydrolize casein. This is the first report on rumen prevascular fungi. Since the facultative anaerobic fungi reported in this study produces useful extracellular digestive enzymes and can colonize and grow a biofilms on the rumen wall they can be used as valuable probiotic in husbandry industry. Keywords : Rumen fungi, Biofilm, Rumen prevasular