Microbiological activity in the natural world is of key importance in the integrated functioning of ecosystems, yet we remain largely ignorant of the role and relevance of the vast majority of microorganisms. This ignorance is largely due to widely acknowledged, but unresolved problems in methodologies. Application of flow cytometry to such studies has already revolutionised our understanding of marine photosynthetic planktonic microorganisms, revealed new levels of complexity in the behaviour of bacterial populations and produced a reliable screening protocol for eukaryotic water-borne pathogens. Advances in fluorescent probe technology now offer realistic approaches for direct cell identification, viability assessment and responses to environmental changes using basic, single light-source flow cytometers. Here we review current applications of flow cytometry in environmental microbiology and present a case for the adoption of the technique as a necessary and routine research instrument.