Deep Hypersaline Anoxic Basin (DHAB) are extreme hypersaline environments located on the seafloor which, despite conditions that are hardly favourable to life, can support thriving ecosystems and demonstrate unique microbial communities. Information on the diversity and functioning of microbial communities living in these harsh environments remains limited, partly because of access to samples and the difficulty of maintaining the sampled microorganisms once back in the laboratory. The brine seawater interface (BSI) is the most active part of DHAB ecosystems and exhibits different successive microbial communities. This study focuses on the Afifi DHAB, a relatively shallow site described in 2020 and located near the eastern coast of the Red Sea, and uses the DNA-Stable Isotope Probing (DNA-SIP) method to link microorganisms’ identity and their associated functions. Seawater layers of different salinities (50 to 250 PSU) from the Afifi BSI were sampled and incubated under anaerobic conditions with different carbon sources: glucose, pyruvate and acetate. Through this approach, the study successfully identified carbon consumers in different layers of the Afifi DHAB and constructed a putative carbon flow among the microorganisms present. Specifically, Vibrionaceae and Halanaerobiaceae are the predominant glucose utilizers, while Thalassospiraceae, Desulfovibrionaceae and Desulfobacteraceae are the main pyruvate and acetate utilizers. Several sulfate-reducing bacteria groups are also identified as potential secondary glucose and pyruvate utilizers, i.e., they utilize the metabolites produced by glucose and pyruvate first consumers. Layers specificities are also revealed, e.g., glucose is consumed by Deferribacteraceae, Rhodobacteraceae and Pseudoalteromonadaceae in the upper, less salty layer, and by Acetothermiia, Melioribacteraceae and Spirochaetacea in the more saline underlying layer. This study provides an initial functional characterization of the microbial community within the Afifi DHAB, highlighting insights into the ecological dynamics and metabolic activities of microorganisms in this unique ecosystem.