Intertidal microphytobenthos (MPB) are a major contributor to primary production in estuarine ecosystems. While their biomass is highly variable at multiple spatial and temporal scales, the underlying drivers are as yet little understood. Both in situ sampling and remote-sensing techniques often lack the temporal resolution or coverage to simultaneously capture short-term (intratidal to daily) and longer-term (weekly to annual) biomass changes. Our field setup with in-situ NDVI sensors allowed us to study MPB surface biomass variability at high temporal resolution (10 mins) for up to two years in a freshwater euglenoid dominated mudflat, and a brackish and a marine diatom dominated mudflat. MPB biomass showed marked periodicities at multiple temporal scales: seasonal, spring-neap and intratidal. The diatom-dominated MPB community showed a seasonal biomass peak in winter, while the euglenoid-dominated community showed biomass peaks during spring and summer, probably caused by underlying divergent responses to mainly irradiance, temperature and wind-induced resuspension, and macrobenthos grazing. Spring-neap periodicity likely resulted from differential migratory responses of the MPB communities to variation in timing and duration of daylight exposure. In the freshwater community, upward migration only occurred when exposure duration was sufficiently long (≥4 h). In the diatom-dominated community, morning daylight exposure resulted in highest NDVI values. This study highlights the differences in MPB biomass dynamics between MPB communities within estuarine ecosystems, and underscores the great potential of high-resolution temporal NDVI monitoring for more accurate estimates of MPB biomass and primary production.