Typical contaminants heavy metals are threatening the survival and health of organisms in the aquatic ecosystem. Therefore, toxic effects and possible mechanisms of five heavy metals, copper (Cu), manganese (Mn), cadmium (Cd), zinc (Zn), lead (Pb), and their mixtures towards Chlorella pyrenoidosa (C. pyrenoidosa) were investigated by the time-dependent microplate toxicity analysis (t-MTA) method. Here, direct equilibration ray method (EquRay) and uniform design ray method (UD-Ray) were used to design ten binary and one quinary mixture systems, respectively. Then, a return-to-zero fitting (RTZF) method was used to analyze toxicity interaction within mixture systems. According to RTZF, one binary and quinary heavy metal mixture systems exhibit time-dependent and component-dependent antagonism. Antagonism in Zn-Cd mixture system increases gradually from 24 to 48 h then decreases gradually from 48 to 96 h. Toxicity interaction within quinary mixture changes from additive action to antagonism and to additive action again over time. The change of chlorophyll-a reduction rate under the action of different heavy metal is slightly different. The toxicity mechanism of copper on algal cells include destruction of cell membrane and chloroplast structure.