Trace elements can be toxic when they cannot be easily removed after entering an ecosystem, so a long-term assessment is fundamental to guide ecosystem restoration after catastrophic pollution. In 1998, a pyrite mining accident in Aznalcóllar (south-western Spain) spilled toxic waste over a large area of the Guadiamar river basin, where, after restoration tasks, the Guadiamar Green Corridor was established. Eight years after the mine accident (2005–2006), the ground-dwelling insectivorous lizard Psammodromus algirus registered high trace-element levels within the study area compared to specimens from a nearby unpolluted control site. In 2017, 20 years after the accident, we repeated the sampling for this lizard species and also quantified trace elements in vegetation as well as in arthropod samples in order to identify remnant trace-element accumulation with the aim of assessing the transfer of these elements through the trophic web. We found remnant trace-element contamination in organisms of the polluted site compared to those from the unpolluted site. All trace-element concentrations were higher in arthropods than in plants, suggesting these compounds bioaccumulate through the trophic web. Lizards from the polluted areas had higher As, Cd, and Hg concentrations than did individuals from the unpolluted area. Lizard abundance between sampling periods (2005–06 and 2017) did not vary in unpolluted transects but strongly declined at polluted ones. By contrast, the Normalized Difference Vegetation Index indicated that in the study period, the vegetation was similar at the two sampling sites. These results suggest that, 20 years after the accident, the trace-element pollution could be the cause of a severe demographic decline of the lizard in the polluted area. Display omitted •Trace elements in three trophic levels were assessed 20 years after a mine spill.•There was an apparent transfer of trace elements from plants to arthropods.•Lizards registered high trace element accumulation for As, Hg, and Cd.•Lizard density strongly declined at the polluted site with respect to the control one.•20 years after the spill, the trace element pollution persists in the Guadiamar basin. 20 years after the mine spill at the Guadiamar basin (southern Spain), plant, arthropod and lizard samples showed high concentration of trace elements, suggesting that the pollution persists.