This study presents results of the first comprehensive research on ecotoxic trace metals (Cd, Pb, Cu and Zn) in aquatic anchialine ecosystems. Data show the influence of hydrological and geological characteristics on trace metals in highly stratified anchialine water columns. Distribution of Cd, Pb, Cu and Zn in two anchialine water bodies, Bjejajka Cave and Lenga Pit in the Mljet National park, Croatia were investigated seasonally from 2006 - 2010. Behaviour and concentrations of dissolved and total trace metals in stratified water columns and metal contents in sediment, carbonate rocks and soil of the anchialine environment were evaluated. Trace metals and dissolved organic carbon (DOC) concentrations in both anchialine water columns were significantly elevated compared to adjacent seawater. Zn and Cu concentrations were the highest in the Lenga Pit water column and sediment. Elevated concentrations of Zn, Pb and Cu in Bjejajka Cave were mainly terrigenous. Significantly elevated concentrations of cadmium (up to 0.3 µg L-1) were found in the water column of Bjejajka cave, almost two orders of magnitude higher compared to nearby surface seawater. Laboratory analysis revealed that bat guano was the major source of cadmium in Bjejajka Cave. Cadmium levels in Lenga Pit, which lacks accumulations of bat guano, were 20-fold lower. Moreover, low metal amounts in carbonate rocks in both caves, combined with mineral leaching experiments, revealed that carbonates play a minor role as a source of metals in both water columns. We observed two types of vertical distribution pattern of cadmium in the stratified anchialine Bjejajka Cave water column. At lower salinities, non-conservative behaviour was characterized by strong desorption and enrichment of dissolved phase while, at salinities above 20, Cd behaved conservatively and its dissolved concentration decreased. Conservative behaviour of Cu, Pb, Zn and DOC was observed throughout the water column. After heavy rains, Cd showed reduced concentration and uniform vertical distribution, suggesting a non-terrestrial origin. Under the same conditions, concentrations of total and dissolved Pb, Cu, Zn and DOC were significantly elevated. Variations of trace metal vertical distributions in anchialine water columns were caused by large inputs of fresh water (extraordinary rainy events), and were not influenced by seasonal changes.