Knowledge of microbial functioning linked to dissolved organic matter (DOM) in hypersaline lakes is limited. This study aimed to understand this relationship in surface and subsurface sediments and how it is affected by daytime and night‐time variability. A sampling regime, including daytime and night‐time measurements, was designed to record aerobic respiration, extracellular enzyme activity (β‐glucosidase, leu‐aminopeptidase and phosphatase), prokaryotic cell density and viability, content of pigment and extracellular polymeric substances (EPS), and organic matter weight in the surface and subsurface sediments. The main physicochemical characteristics and optical properties of DOM in the surface and interstitial waters were determined. Larger differences were detected in biofilm microbial activity between surface and subsurface sediments than between day and night, as a result of higher prokaryotic cell density and higher availability of fresh DOM at the surface, possibly released from primary production. Redox conditions, pH and DOM quantity and quality were strongly correlated with enzyme activity and variability in respiration, and might determine (through modifying nitrogen (N) and phosphorus (P) availability) how microbes use organic compounds differently at the two sediment depths. Low prokaryotic viability indicated that the microbial community was stressed in both surface and subsurface sediments. By contrast, higher EPS and pigment content and lower metabolic efficiency at the surface layer indicated harsher conditions compared to the subsurface layer. Pigment presence and variation in daily oxygen levels in the subsurface layer indicate that the two sediment depths are interconnected. Results suggest that surface and subsurface microbes have a distinct role in hypersaline shallow lake sediment. Microorganisms in surface sediments form a structured mat with high EPS and carotenoids and enhanced carbon metabolism. The mat also serves as a protection shield from light radiation, salinity and desiccation. At the subsurface, the lower availability of fresh organic compounds and oxygen reduces microbial biomass and activity which is, in turn, more efficient and focused on N and P acquisition.