The performance of pressure-driven membrane filtration processes for water treatment is hampered by biofouling. A relevant, but often overlooked aspect of this phenomenon concerns the localized microenvironment at the membrane interface. A key question is the level of stress on adhering cells and how this impacts on the developing biofilm. In this study, Pseudomonas fluorescens biofilms were monitored after 1, 2 and 7-day crossflow nanofiltration experiments using confocal microscopy with live/dead staining which enabled analysis of both biofilm structure and the spatial localization of dead versus live cells. A significant increased level of biomass at low-compared to high-flux conditions (2-day experiments) suggested hindrance of bacterial proliferation at higher fluxes. An increase in live cell fractions was generally observed between 24-and 48-h at low flux conditions (3 bar), while the fraction of dead/injured cells remained constant during that same period. At higher flux conditions (15 bar), the volume of live cell fractions remained constant over 24-and 48-h experiments. The implications of these findings point to the need to reevaluate classical contact-killing strategy for controlling membrane fouling; initial membrane fouling events are characterized by an initially-induced cell death stage followed by an adaptation period through which surviving cells are able to acclimatize in their respective environments. This study emphasizes the need to better understand the role of operating parameters and its resulting cell death during early stage fouling. It is in this context that fouling management strategies can be further developed.