Aquatic ecosystems face variable exposure to pesticides, especially during floodings which are associated with short bursts of high contaminant concentrations that influence biological systems. A study was undertaken to highlight the impact of the herbicide diuron applied in mixture with the fungicide tebuconazole on natural periphyton during flooding events. Periphyton were grown in two series of two lotic outdoor mesocosms: one series was non-contaminated (control) while the other was exposed to chronic contamination. One channel of each series was exposed to pulses of the tested pesticides. After 4 weeks, these two channels were exposed to three successive pulses, with each pulse followed by one week of recovery. Impacts on periphyton were assessed by using Denaturing Gel Gradient Electrophoresis (DGGE) to measure community structure. At a functional scale,photosynthetic efficiency was quantified during each pulse, and the induced tolerance (PICT)to diuron was estimated. Moreover, pesticide oncentrations in the water column and periphyton matrix were measured. Diuron was adsorbed in the periphyton during each pulse and desorbed 13 h after pulse end. During the first pulse, photosynthetic efficiency was correlated with pesticide concentration in the water phase, and there was no difference between periphyton from chronically contaminated channels and control channels.However,during the second and third pulses, the Photosynthetic efficiency of periphyton chronically exposed to pesticides appeared to be less impacted by the acute pulsed exposure of pesticide.These changes were consistent with the acquisition of induced tolerance to diuron. Our experimental study indicates that the effects of pulsed acute exposures to pesticides on periphyton depended on whether the communities had previously been exposed to the same stressors or not. Thus, pulsed and chronic exposures cannot be considered separately for assessing the effects of pollutants on non-target organisms in lotic systems, since in situ hydrological dynamics combine both base-flow (chronic) and flood (pulse) periods.