Seine river water was used as natural environmental medium to study the ecotoxicological impact of ZnO and CdS nanoparticles and Zn2+ and Cd2+ free ions using Chlorella vulgaris as a biological target. It was demonstrated by viability tests and photosynthetic activity measurements that free Zn2+ (IC50 = 2.7 × 10−4 M) is less toxic than free Cd2+ and ZnO nanoparticles (IC50 = 1.4 × 10−4 M). In the case of cadmium species, free Cd2+ (IC50 = 3.5 × 10−5 M) was similar to CdS nanoparticles (CdS-1: IC50 = 1.9 × 10−5 M and CdS-2: IC50 = 1.9 × 10−5 M), as follows: CdS > Cd2+ > ZnO > Zn2+. Adenosine-5’-triphosphate (ATP) assay and superoxide dismutase (SOD) enzymatic activity confirmed these results. Transmission electron microscopy (TEM), coupled with energy-dispersive X-ray spectroscopy (EDS), confirmed the internalization of CdS-1 nanoparticles after 48 h of contact with Chlorella vulgaris at 10−3 M. With a higher concentration of nanoparticles (10−2 M), ZnO and CdS-2 were also localized inside cells.