Aims: To develop a rapid and simple method for the assessment of metabolic activity of bacteria in natural environment. Methods and Results: A rapid and simple multicolour digital image analysis system for enumerating viable bacteria based on active fluorescent staining has been developed. This system can accurately differentiate actively respiring bacteria and non-respiring bacteria by distinctive colour information in the digital image captured by an epifluorescence microscope equipped with low magnification objective lens. An algorithm to distinguish bacteria from considerable detritus, which produced bright fluorescence in different colours, by colour segmentation has also been developed. This system was applied to river water samples, and the total and respiratory active bacterial counts by digital image analysis were highly related to those by epifluorescence microscopy (r(2) = 0.96 and 0.93, respectively). Conclusion: This system allowed the rapid and simple differentiation of bacteria from detritus and concurrent assessment of their metabolic activity, with results being available within 1 h. Significance and Impact of the Study: The low magnification image analysis allows more rapid and simple quantification of bacteria and could considerably decrease image data amount and acquisition time. This system could be easily applied to the rapid analysis of microbes in various environments.