The Yucatan Peninsula is one of the world’s largest karstic aquifer systems. It is the sole freshwater source for human users and ecosystems. The region hosts internationally important groundwater-dependent ecosystems (GDEs) in the 5280km2 Sian Ka’an Biosphere Reserve. The GDEs are threatened by increasing groundwater abstractions and risks of pollution. Hydrogeological exploration work is needed as basis for sound groundwater management. A multidisciplinary approach was used to study this data-scarce region. Geochemical data and phreatic surface measurements showed distinct hydrogeological units in the groundwater catchment of Sian Ka’an. The hilly southwestern areas had a low hydraulic permeability, likely caused by a geology containing gypsum, whereas the transition zone and flat areas in the east and north had a high permeability. In the latter areas, the fresh groundwater could be described by a Dupuit–Ghyben–Herzberg lens. Geophysical borehole logging and time-domain electromagnetic soundings identified a shallow, low-resistive and high-gamma-radiation layer present throughout the hilly area and transition zone. Its thickness was 3–8m, apparent conductivity was 200–800mS/m and natural gamma-radiation about 80 counts pr. second. The layer is proposed to be ejecta from the Chicxulub impact (Cretaceous/Paleogene boundary). Spatial estimates of recharge were calculated from MODIS imagery using the ‘triangle method’. Average recharge constituted 17% of mean annual precipitation in the study area. Recharge was greatest in the hilly area and towards Valladolid. Near the coast, average actual evapotranspiration exceeded annual precipitation. The multidisciplinary approach used in this study is applicable to other catchment-scale studies.