Photosynthesis and respiration were analyzed in natural biofilms by use of O2 microsensors. Depth profiles of gross photosynthesis were obtained from the rate of decrease in O2 concentration during the first few seconds following extinction of light, and net photosynthesis of the photic zone was calculated from O2 concentration gradients measured at steady state. Respiration within the photic zone was calculated as the difference between gross and net photosynthesis. Two types of biofilms were investigated: one dominated by diatoms, and one dominated by cyanobacteria. High O2/CO2 ratios caused increased respiration especially within the diatom biofilm, which could indicate that photorespiration was a dominant O2‐consuming process. The rate of respiration was constant within both biofilms during the first 4.6 s following extinction of light, even when respiration was stimulated by high O2/CO2 ratio. The assumption of a constant rate of respiration during the dark period is an essential one for the determination of gross photosynthetic activity by use of O2 microsensors. We here present the first evidence to substantiate this assumption. The results strongly suggest that gross photosynthesis as measured by use of O2 microsensors may include carbon equivalents that are subsequently lost through photorespiration. Computer modeling of photosynthesis profiles measured after 1.1, 1.6, and 2.6 s of dark incubation illustrated how the actual photosynthesis profile could have appeared if it had been possible to do the determination at time 0. Diffusion of O2 during the up to 4.6‐s long dark incubations did not affect gross photosynthetic rate when integrated over all depths, but the apparent vertical distribution of the photosynthetic activity was strongly affected.