Estuaries may be reservoirs of a wide variety of pollutants, including mutagenic and carcinogenic substances that may impact on the ecosystem and human health. A previous study showed that exposure of human hepatoma (HepG2) cells to extracts from sediment samples collected in two areas (urban/industrial and riverine/agricultural) of an impacted estuary (Sado, Portugal), produced differential cytotoxic and genotoxic effects. Those effects were found to be consistent with levels and nature of sediment contamination. The present study aimed at evaluating whether the mixtures of contaminants contained in those extracts were able to modulate DNA repair capacity of HepG2 cells. The residual level of DNA damage was measured by the comet assay in cells exposed for 24 or 48 h to different extracts, after a short preexposure to a challenging concentration range of ethyl methanesulfonate (EMS), as a model alkylating agent. The results suggested that the mixture of contaminants present in the tested samples, besides a potential direct effect on the DNA molecule, may also interfere with DNA repair mechanisms in HepG2 cells, thus impairing their ability to deal with genotoxic stress and, possibly, facilitating accumulation of mutations. Humans are environmentally/occupationally exposed to mixtures rather than to single chemicals. Thus, the observation that estuarine contaminants induce direct and indirect DNA strand breakage in human cells, the latter through the impairment of DNA repair, raises additional concerns regarding potential hazards from exposure and the need to further explore these endpoints in the context of environmental risk assessment.