The extensive use of zinc oxide nanoparticles (ZnO NPs) in cosmetics, sunscreens and healthcare products increases their release in the aquatic environment. The present study explored the possible interaction of ZnO NPs with montmorillonite clay minerals in aqueous conditions. An addition of ZnO NPs on clay suspension significantly (p < 0.05) increases the hydrodymic size of clay particles from 1652 ± 90 nm to 2158 ± 13 nm due to heteroagglomeration. The electrokinetic measurements showed a significant (p < 0.05) difference in the electrophoretic mobilities of bare (−1.80 ± 0.03 μm cm/Vs) and ZnO NPs-clay association (−1.37 ± 0.03 μm cm/Vs) that results to the electrostatic interaction between ZnO NPs and clay particles. The attenuated total reflectance Fourier transform infrared spectroscopy analysis of ZnO NPs-clay association demonstrated the binding of ZnO NPs with the Si-O-Al region on the edges of clay particles. The increase in size of ZnO NPs-clay heteroagglomerates further leads to their sedimentation at 24 h. Although, the stability of ZnO NPs in the clay suspension was decreased due to heteroagglomeration, but the bioavailability and toxicity of ZnO NPs-clay heteroagglomerates in Tetrahymena pyriformis was enhanced. These observations provide an evidence on possible mechanisms available in natural environment that can facilitate nanoparticles entry into the organisms present in lower trophic levels of the food web.