The fungicide myclobutanil (MYC) is a common contaminant found in surface water. The aim of this study was to determine the acute toxicity, developmental effects, bioconcentration factor (BCF) and potential bio-molecular mechanisms of MYC toxicity in zebrafish. Susceptibility to MYC toxicity was life-stage dependent with adult fish being the most sensitive (96 h-LC50, 6.34 mg/L) followed by 72 h post-hatch (hph) larvae (8.90 mg/L), 12 hph larvae (20.53 mg/L) and embryos (42.54 mg/L). Zebrafish embryos and larvae (12 hph) responded with decreased hatching, heartbeat and growth, as well as abnormal spontaneous movement and development. BCFs were calculated by quantifying MYC concentrations from different tissues of adult zebrafish exposed to MYC for up to 11 days. Highest BCFs were obtained from gills (18.25 ± 0.07), followed by viscera (16.78 ± 0.04), head (13.13 ± 0.08) and muscle (8.96 ± 0.10). MYC (0.5 mg/L) inhibited gene expression related to cholesterol synthesis pathway, including 24-dehydrocholesterol reductase (DHCR24), 7-dehydrocholesterol reductase (DHCR7), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCRa), HMGCRb, farnesyl-diphosphate farnesyltransferase 1(FDFT1), squa-lene epoxidase (SQLE), isopentenyl-diphosphate delta isomerase 1 (IDI1) and CYP51, while no cholesterol changes were observed in the MYC treated group. These results will contribute to the literature assessing the environmental risk of MYC in aquatic environment.