Microalgae are widely used as models for ecotoxicological assays. The present study investigated the physiological responses of Chlorella vulgaris to five days of exposure to copper and zinc at different concentrations (control, 125, 250, 500 and 1,000 µM). Both heavy metals showed dose-dependent cellular accumulation. Decreased maximum quantum efficiency of photosystem II (Fv/Fm) was influenced by both heavy metal concentration and time of exposure. A reduction in Fv/Fm indicated that photodamage occurred starting from day 3 of exposure. Other toxicity symptoms included chlorophyll degradation and an increase in reactive oxygen species (ROS). While exposure to both heavy metals resulted in a decrease in chlorophyll a content to a similar extent, an increase in ROS was detected only for 1,000 µM copper, suggesting stronger toxicity effects of copper compared to zinc. Nevertheless, an increase in lipid peroxidation was not detected, indicating that ROS produced by 1,000 µM copper was not sufficient to induce disintegration of membrane lipids via the oxidation process. Proline, an amino acid with various putative protective functions against stress, exhibited a rapid increase with increases in heavy metal concentration and time of exposure. These results provide a set of effective biomarkers for heavy metal contamination using C. vulgaris as a bioindicator.