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Abstract Pulses of resource availability along environmental gradients can filter the local and regional distribution of macrophyte and microbial mat communities in wetlands. Wetlands that experience short hydroperiods (i.e., <6 months with standing water) may cause macrophyte and microbial mat competition for water. However, the stress gradient hypothesis predicts that abiotic stress should increase facilitative co‐regulation of producer dynamics. To determine if and how macrophyte and microbial mat biomass covary along a hydrologic gradient, we conducted two observational surveys and a biomass removal experiment in Everglades National Park, FL, USA. In the survey, macrophyte and microbial mat biomass were measured over a two‐year period across nine hydrologically regulated macrophyte community types to determine drivers of biomass and macrophyte–microbial mat interactions along a hydroperiod gradient (3–8 months) using a structural equation model. In the experiment, the effect of hydrology on the interaction between macrophytes and microbial mats was quantified by measuring the effect of bimonthly removal of macrophyte or microbial mat biomass on the biomass of both communities in plots in wetlands with contrasting hydroperiods (3–6 months). Hydrology and biological interactions influenced macrophyte and microbial mat biomass, with stronger interactions observed in the shortest hydroperiod transect sites dominated bySchoenus nigricansandCladium jamaicense. Along the hydrologic gradient, we found direct negative effects of macrophyte biomass on microbial biomass and vice versa, and a significant positive effect of microbial response to flooding duration on macrophyte biomass. Experimental macrophyte removal in shorter‐hydroperiod wetlands resulted in a significant increase in microbial biomass while microbial mat removal reduced biomass of the dominant macrophyteC. jamaicense. The facilitative effect of microbial mats on macrophyte biomass in shorter‐hydroperiod wetlands may be driven by mats prolonging soil moisture retention due to their desiccation‐resistant structure. Stress‐induced facilitation supported the stress gradient hypothesis across the short‐hydrologic gradient, while competitive interactions were also observed. As climate and human drivers continue altering hydrology in aquatic systems, the type and strength of community interactions will continue to shift and alter distributions across the landscape.