Microbial mats and microbialites are essential tools for reconstructing early life and its environments. To better understand microbial trace element cycling, a microbial mat was collected from the sinkhole systems of the western shores of the Dead Sea, a dynamic environment exhibiting diverse extreme environments. Intense arsenic enrichment (up to 6.5 million times higher than current water concentrations, and 400 times the bulk concentration in the mat) was measured. Arsenic was dominantly found as As(V) in organic molecules, as shown by XANES spectra and high-resolution elemental mapping. Arsenic cycling genes obtained from metagenomic analysis were associated with arsenic detoxification, supporting an active mechanism of As(V) uptake, As(III) efflux and organo-arsenic accumulation in microbial mat extracellular polymeric substances. Thus, we propose that such localized enrichment of As can be attributed to a transient increase in As(V) concentrations in the circulating subsurface water of the Dead Sea shore and its subsequent incorporation in organoarsenic molecules through microbial detoxification processes. Our dataset supports the possibility of metalloid enrichments recorded in very localized facies due to rapid geogenic fluctuations in chemistry of the water flowing over a biofilm. In this context, this example calls for caution when interpreting metal(loid) enrichment in organic matter-rich layers and microbialites of Paleoproterozoic origins. Arsenic signatures in Precambrian organic matter and carbonate rocks may host biosignatures, including evidence of extracellular polymeric susbtances, As-binding and detoxification processes, without supporting arsenotrophy. They do, however, provide clues to better assess paleoenvironmental conditions at the time of microbial mat formation and sedimentation.