This dataset originates from a laboratory mesocosm experiment investigating the effects of simulated marine heatwaves (MHWs) on benthic microbial communities from sublittoral sediments of Spiekeroog (Wadden Sea, Germany). Surface sediments were sieved (1 mm) and incubated under controlled light and temperature conditions before exposure to three temperature treatments (19 °C control, 23 °C, and 25 °C). Following a gradual warming phase, MHW conditions were maintained for six days, followed by a recovery phase at ambient temperature. Sediment samples were collected repeatedly throughout the experiment. Surface sediments (upper 2 cm) were collected using syringe cores. Pooled samples were analyzed for chlorophyll a as a proxy for microphytobenthos biomass using ethanol extraction and spectrophotometric pigment analysis. Extracellular polymeric substances (EPS) were quantified using EDTA extraction followed by phenol–sulfuric acid carbohydrate analysis. DNA was extracted from sediment subsamples using a Qiagen PowerSoil kit. Prokaryotic abundance was estimated by quantitative PCR targeting the 16S rRNA gene (primers 519F/907R), using an Escherichia coli 16S rRNA gene standard curve. The dataset includes chlorophyll a concentrations (µg g⁻¹ dry sediment), EPS carbohydrate concentrations, and prokaryotic 16S rRNA gene copy numbers for all sampling times, elevations, and treatments.