The malarial parasite Plasmodium falciparum has evolved resistance to several antimalarial drugs, posing a significant challenge to the effective management and treatment of malaria in endemic areas. Drug repurposing has emerged as a potential alternative strategy for addressing this issue. This study aimed to identify an FDA-approved microbicidal agent, zinc pyrithione (ZPT), against mixed blood-stage parasites of P. falciparum chloroquine-sensitive (Pf3D7) and resistant strains (PfINDO). Based on the time-inhibition kinetics assay, the parasite viability was significantly inhibited by ZPT treatment for 96 h (0.77 μM and 0.37 μM) and 72 h (0.63 μM and 0.61 μM), followed by 48 h (0.76 μM and 1.32 μM) and moderate inhibitory effects for 12 and 24 h in both Pf3D7 and PfINDO culture. Stage-specific treatment revealed that trophozoites and schizonts exposed to ZPT were more susceptible than ring-stage parasites. Phenotypic assays revealed that trophozoites and schizonts failed to mature and exhibited aberrant morphologies such as condensed nuclei, as determined by Giemsa staining. Furthermore, ZPT in combination with dihydro-artemisinin and chloroquine demonstrated additive interactions in both Pf3D7 and PfINDO parasites. At therapeutic dosages, ZPT failed to cause hemolysis in human erythrocytes. Overall, this study demonstrated a time-dependent effect of ZPT on the blood stages of human P. falciparum in culture, suggesting its utility in clinical settings.