Resuspension thresholds in terms of friction velocity were experimentally quantified for the prokaryotes, protists and for the first time, viruses of intertidal mudflat biofilms. Differences in resuspension thresholds could be related to the type, behaviour and size of microorganisms and their association with particles. Free microorganisms (viruses, bacteria and some nanoflagellates) were resuspended by weak flow at friction velocities lower than 2 cm s− 1. Chlorophyll a, some nanoflagellates and attached bacteria were resuspended together with the bed's muddy sediment, which required friction velocities larger than 3 cm s− 1. Diatoms smaller than 60 μm were resuspended at velocities between 3 and 5 cm s− 1, while those larger than 60 μm were resuspended at higher friction velocities (5.5 to 6.5 cm s− 1). The thresholds of resuspension also depended on the micro-scale position of microorganisms in the sediment (horizontal and vertical distributions). In the field, the vertical distribution of chlorophyll a (a proxy of microphytobenthos) was skewed, with a maximum in the first 2 mm of sediment. Along the neap–spring tidal cycle, chlorophyll a revealed an increase in MPB biomass in the first 2 mm of the sediment, in relation to light increases with exposure durations. The horizontal distribution of chlorophyll a could be inferred from erosion experiments. During the initial phase of biofilm growth, the distribution of chlorophyll a seemed horizontally homogeneous, and was uniformly eroded at the beginning of the increase in chlorophyll a. From these results, we can make a hypothesis: in the subsequent phase of biofilm growth until the maximum of emersion duration, the eroded quantity of chlorophyll a was larger than expected based from chlorophyll a vertical distribution, suggesting that biofilm horizontal distribution became patchy and enriched chlorophyll a was preferentially eroded. When emersion duration and biofilm growth decreased, the trend was reversed, and eroded quantity of chlorophyll a was lower than expected from chlorophyll a vertical distribution, suggesting that areas with low chlorophyll a were preferentially eroded. Such erosion patterns when biofilm growth decreased probably resulted from the bulldozing activity of a surficial sediment bioturbator, the gastropod Peringia ulvae. Our study did not directly prove this horizontal distribution but it should be further discussed. This distribution needs to be studied to acquire real evidence of patchy distributions.