The study evaluated the selective inhibition method for measuring the fungal and bacterial distribution in the activated sludge, using glucose as sole carbon source, and cycloheximide and streptomycin as bactericide and fungicide, respectively, substrate-induced oxygen uptake rate (OUR) as bioactivity index. Three samples were examined, i.e., aerobic sludge in a wastewater treatment plant, soils and anaerobic sludge. As selective microbial biocides were applied, the OUR of aerobic and soils sludge reduced, whereas the OUR of anaerobic sludge was stimulated. When 1 mg/g cycloheximide and 2 mg/g streptomycin were applied, the inhibition index D of aerobic sludge was approximately 1; however, the values of D for soils were greater than 1 at all the dosages of bactericide or fungicide applied in this study. These results suggested that the antibiotics used in this study could selectively inhibit aerobic sludge and partially inhibit the soils. It was found that the selective inhibition method was suitable for aerobic sludge analysis, but encountered problems for the other samples, e.g., failure inhibition effect of antibiotic, partial inhibition and low accuracy. Further studies are therefore needed in the selection of antibiotic and microbial activity parameter.