Microstructural examinations were performed on the anaerobic biofilm from reactor filled with PE support media. Optical microscope, SEM and fluorescent microscope were used for qualitative and morphological studies on the attached microorganism under anaerobic condition. Microorganisms were attached in crevices where protection from shear forces of surfaces where easy to contact with support media surface. A hypothesis for biofilm accumulation occurs on a surface such as polymer support media is presented schematically : 1st step ; cell-support media attachment, 2nd step ; cell-support media attachment and cell-cell attachment, 3rd step ; attached biofilm from neighboring crevices joins together and growing, 4th step ; mature and irregualar biofilm was formed. In SEM photographs, shape and structures of biofilm were observed, but microorganism species and methanogens were not identified. A large number of methanogenic bacteria were identified on the surface of PE substratum by fluorescence under 480nm of radiation and it was estimated that methanogenic bacteria was related to initial attachment of bacteria under anaerobic condition.