Intermittent water flow regimes characterize streams in many world regions, especially those with arid and semiarid climates. During cease to flow conditions, biofilms on streambed sediments may be exposed to desiccation. Environmental conditions and resource availability change with desiccation and may influence biofilm functioning and whole stream ecosystem processes. Rainfall events during the nonflow phase can rehydrate streambed sediments, but the effect of these pulses on biofilm functioning is unclear. This study aimed to analyze the effects of a rehydration event on biofilm functional diversity during the nonflow period in a subtropical Australian stream. Biofilms from three different stream pools on the same reach; one permanently water-covered and the other two differing in their desiccation time were studied. Biofilms initially differed owing to the time they were exposed to dry conditions but rehydration events significantly increased biofilm functional diversity, producing a “reset” effect on the desiccation exposure, as after that bacterial functioning decreased again because of the new dry conditions. The observed rapid biofilm responses to rehydration during flow intermittency might be essential in sustaining biofilm functional diversity in intermittent streams.