Previous studies on copper pyrithione (CPT) and zinc pyrithione (ZPT) as antifouling agents have mainly focused on marine organisms. Even though CPT and ZPT pose a risk of human exposure, their neurotoxic effects remain to be elucidated. Therefore, in this study, the cytotoxicity and neurotoxicity of CPT and ZPT were evaluated after the exposure of human SH-SY5Y/astrocytic co-cultured cells to them. The results showed that, in a co-culture model, CPT and ZPT induced cytotoxicity in a dose-dependent manner (~ 400 nM). Exposure to CPT and ZPT suppressed all parameters in the neurite outgrowth assays, including neurite length. In particular, exposure led to neurotoxicity at concentrations with low or no cytotoxicity (~ 200 nM). It also downregulated the expression of genes involved in neurodevelopment and maturation and upregulated astrocyte markers. Moreover, CPT and ZPT induced mitochondrial dysfunction and promoted the generation of reactive oxygen species. Notably, N-acetylcysteine treatment showed neuroprotective effects against CPT- and ZPT-mediated toxicity. We concluded that oxidative stress was the major mechanism underlying CPT- and ZPT-induced toxicity in the co-cultured cells.