In 2001–2002, two ground collapses occurred in the island of Nisyros (Aegean Sea, Greece), which formed a 600 m long and up to 5 m wide fissure in the vegetated central part of the Lakki Plain caldera. The fissure was alternatively ascribed to tensional stress release and hydrothermal alteration. In this study, we present original data of diffuse CO2 soil fluxes, soil temperatures, mineralogical and chemical composition of the caldera-filling deposits exposed on the fissure walls, and chemical and isotopic composition of interstitial soil gases collected from: the bottom of the fissure, the adjacent vegetated areas, the hydrothermal craters, and selected sites outside the caldera. The occurrence of intense hydrothermal alteration was shown by both mineralogical and chemical analyses of the fissure walls material. Typical mineral assemblage and enrichments in incompatible elements related to advanced argillic alteration, characterizing steam-heated hydrothermal environments, were recognized. Although the low-permeable sediment cover in the Lakki Plain concealed the underneath hydrothermal gas flow, preventing anomalous soil temperatures and CO2 fluxes, the chemical and isotopic composition of the interstitial soil gases revealed an active hydrothermal fluids circulation below the collapsed area, likely controlled by buried structural lineaments. Hydrothermal alteration can then be invoked as the most likely trigger mechanism for the 2001–2002 collapse event.