Microbial colonization of the different support materials used to enhance methane production in anaerobic digesters is rapid and occurs in the first 24 h of sludge incubation. Scanning electron microscopy studies reveal a predominant presence of filamentous methanogenic forms, closely resembling Methanosaeta (Methanothrix), which are located on the outer layer and in the bacterial framework of the biofilm. These findings are consistent with the results obtained from microbial counts using both the most probable number and epifluorescence microscopic techniques, which show an increase in the numbers of aceticlastic methanogens compared to other microbial groups involved, such as sulphate-reducing bacteria, the numbers of which are similar to those obtained under the initial conditions. Moreover, a sharp increase in the bacterial counts is observed by using the epifluorescence microscopic technique applied to homogenized samples, probably due to the count of bacteria released from the support materials.