The respiratory quinone profile was successfully used as a tool for identifying the change of bacterial phase in the microbial film of aerobic submerged biofilter treating refractory chemicals such as dimethylformamide (DMF). It was ascertained that the coexistence of easily biodegradable substances such as glucose and peptone hindered the acclimation of bacteria inhabiting the microbial film in biofilter to DMF. The higher initial microbial concentration and the feeding of DMF as a sole organic substance accelerated the acclimation of microbial film to DMF. Appreciable changes in quinone profile were detected through the acclimation to DMF, and the results showed that DMF degrading bacteria such as Methylobacterium and Mycobacterium had grown in the biofilter.