The changes in physical properties and microbial activities were investigated during initial biofilm formation in lab-scale anaerobic fluidized bed reactors. Four different upflow velocities, i.e., 4, 7, 14 and 25 m·hr−1 were applied to four respective reactors of an equal size. The upflow velocities caused a prominent difference in the pattern of initial biofilm formation. The biofilm thickness attained eventually approximately 100 μm after 100 days of operation, independent of upflow velocity. On the contrary the biofilm density varied from 4.4 to 24.1 mg-VSS·cm−3 with an increase in the upflow velocity imposed. The activity of acetoclastic methane production increased remarkably 15 to 30 fold of seed sludge, regardless of upflow velocity. Microbial activities with respect to acetate production, H2-utilizing methanogenesis and acetate-utilizing methanogenesis increased finally up to 3-4 times as large as those of suspended grown sludge in a chemostat.