MOSE is a system of mobile gates engineered to temporarily isolate the Venice Lagoon from the Adriatic Sea and to protect the city from flooding during extreme high tides. Within the framework of the Venezia2021 program, we conducted two enclosure experiments in July 2019 (over 48 h) and October 2020 (over 28 h) by means of 18 mesocosms, in order to simulate the structural alterations that microphytobenthos (MPB) assemblages might encounter when the MOSE system is operational. The reduced hydrodynamics inside the mesocosms favored the deposition of organic matter and the sinking of cells from the water column towards the sediment. Consequently, MPB abundances increased over the course of both experiments and significant changes in the taxonomic composition of the community were recorded. Species richness increased in summer while it slightly decreased in autumn, this latter due to the increase in relative abundances of taxa favored by high organic loads and fine grain size. By coupling classical taxonomy with 18S rRNA gene metabarcoding we were able to obtain a comprehensive view of the whole community potential, highlighting the complementarity of these two approaches in ecological studies. Changes in the structure of MPB could affect sediment biostabilization, water turbidity and lagoon primary production.