Aims Interactions with water bacteria affect the incorporation of pathogens into biofilms and thus pathogen control in drinking water systems. This study was to examine the impact of static vs flow conditions on interactions between a pathogen and a water bacterium on pathogen biofilm formation under laboratory settings. Methods and Results A pathogen surrogate Escherichia coli and a drinking water isolate Stenotrophomonas maltophilia was selected for this study. Biofilm growth was examined under two distinct conditions, in flow cells with continuous medium supply vs in static microtitre plates with batch culture. E. coli biofilm was greatly stimulated (c. 2–1000 times faster) with the presence of S. maltophilia in flow cells, but surprisingly inhibited (c. 65–95% less biomass) in microtitre plates. These divergent effects were explained through various aspects including surface attachment, cellular growth, extracellular signals and autoaggregation. Conclusions Interactions with the same water bacterium resulted in different effects on E. coli biofilm formation when culture conditions changed from static to flow. Significance and Impact of Study This study highlights the complexity of species interactions on biofilm formation and suggests that environmental conditions such as the flow regime can be taken into consideration for the management of microbial contamination in drinking water systems.