Understanding how microorganisms adapted to the high arsenic concentration present on early Earth requires understanding of the processes involved in the arsenic biogeochemical cycle operating in living microbial mats. To this end, we investigated a living microbial mat from Laguna Brava (Salar de Atacama, Chile), a hypersaline lake with high arsenic concentration, using an array of conventional geochemical techniques, such as X-ray diffraction, SEM-EDX and Confocal Laser Scanning Microscopy (CLSM), combined with state-of-the-art high resolution scanning imaging techniques, including X-ray micro-fluorescence (mu XRF) and X-ray Absorption Near Edge Structure (XANES) mapping. This experimental approach allowed us to unravel the relationship between the microbial mat activity, mineral occurrence, arsenic speciation, distribution of major and trace elements and their relationship with the mineralogy and the exopolymeric substances (EPS). We show that As was not linked to Ca or Si, and only moderately related to Fe, resulting from sorption onto an iron (oxy)hydroxide mineral. In addition, we were able to identify organic-rich globules containing significant As but no other trace metal(loid)s, and determine the co-existence of As(III) and As(V). These observations strongly support the occurrence of microbially-mediated arsenic cycling in these microbial mats.