In this study, the enumeration of yeast and bacterial cells was investigated using fluorescent in situ hybridization (FISH) in combination with flow cytometry method (FCM) after disruption of pseudomycelia or flocs by sonication. Firstly, mixed yeast samples with developed pseudomycelia and mixed bacteria samples, i.e. activated sludge flocs, were sonicated and investigated using the FCM following the staining with fluorescent dyes. The optimal sonication condition for both biological samples was 60-90 s at 100W. Excessive sonication treatment, i.e. for 120 s, had different effects on pure cultures of bacteria (or yeasts) and mixed samples of bacteria (or yeasts). Subsequently, the simultaneous enumeration of yeasts and bacteria in binary samples was investigated by FISH-FCM using two probes (PF2、EUB338). The binary samples were prepared using Candida tropicalis and Escherichia coli as model yeast and bacteria microorganisms, respectively, and contained 107 cells/ml of background microorganisms and varied concentrations of the target microorganisms ranging from 102-107 cells/ml. Flow cytometry could clearly distinguish yeasts, bacteria and background microorganisms, and one injection provided enumeration results for both target microorganisms simultaneously. FISH-FCM effectively measured concentrations of the target microorganisms as low as 104 cells/ml for yeasts and 103 cells/ml for bacteria, corresponding to 1% and 0.1%, respectively. However, at concentrations < 104 cells/ml for yeasts and < 103 cells/ml for bacteria, the target microorganisms could not be accurately enumerated due to the presence of background microorganisms in the binary sample.