The co-exposure of soil organisms to ZnO nanoparticles (ZnO NPs) and pesticides is likely to take place in agricultural soils. However, the impacts of co-exposure on terrestrial ecosystems are virtually unknown. In this paper, Eisenia andrei was exposed for a 28-day period to serial concentrations of ZnO NPs and/or the organophosphate insecticide chlorpyrifos (CPF) in natural soil, and was evaluated for single and joint effects. Zn and CPF accumulation in earthworm tissue was also determined. In the single assay, ZnO NPs and CPF caused statistical significant effects on survival and growth, but mainly on reproduction. Significant reductions in fecundity and fertility were detected with EC50 values of 278 and 179 mg Zn/kg for ZnO NPs, and of 50.75 and 38.24 mg/kg for CPF, respectively. The most notable effect on biomarkers was the reduction in acetylcholinesterase (AChE) activity caused by CPF, which reflected the neurotoxicity of this compound. The results of the combined assay indicated that co-exposure to ZnO NPs and CPF increased adverse effects in E. andrei. According to the independent action model, the binary mixtures showed a synergism (a stronger effect than expected from single exposures) on earthworm reproduction, which became up to 84% higher than the theoretically predicted values. Zn, and especially CPF accumulation, were influenced by the co-exposure. These results underpin the need to consider the effects of mixtures of NPs and organic chemicals on soil to adequately make ecological risk assessments of NPs. Display omitted •Interaction of ZnO NPs with chlorpyrifos (CPF) in soil can modify intrinsic toxicity.•Co-exposure moduled Zn, and increased CPF accumulation in earthworms.•Joint exposure showed synergistic effects on earthworm reproduction.•Co-exposure studies in soil are necessary for ecological risk assessments of NPs.