The direct effects of pesticides on aquatic bacteria are poorly known. We experimentally investigated the direct effects of diuron (herbicide) on the composition and activities of lake bacterioplankton, using Denaturing Gradient Gel Electrophoresis (DGGE), cloning/sequencing, and flow cytometry with dyes that allow detection of dead cells, cells with depolarized membranes and cells with esterase activity (for physiological state). Generally, diuron had negative impacts on bacterial viability and abundance. Bacterial production strongly correlated with ammonium in controls, but not in diuron-treated samples. Moreover the increase in nitrate concentration with the proportion of dead bacteria was significantly higher in controls, providing evidence not previously shown for natural communities, that diuron may alter the mineralization of organic matter and nitrification. A picocyanobacteria and members of the family Flavobacteriaceae, known to degrade complex polymeric organic matter in aquatic systems were negatively affected by diuron. Except that, the DGGE banding patterns in controls and in polluted samples were generally similar, suggesting no perceptible susceptibility of major bacterial groups, and contrasting with previous reports that diuron has a strong impact on bacterial community composition. Our data suggest that diuron may affect functioning of aquatic systems through negative impacts on some bacterial phylotypes and bacterial cycling of nitrogen.