This study focuses on the potential hazards of borneol (BO) to aquatic organisms and human health. BO has antibacterial, anti-inflammatory and antioxidant activities, and is widely used in medicine, cosmetics, and detergents. In this study, zebrafish was used as a model organism to systematically evaluate the effects of BO on the heart, liver, kidney, and nervous system. The effects of BO on metabolites of zebrafish were studied using MALDI-MSI. The results showed that a high concentration of BO (500 μM) could induce morphological abnormalities (swim-bladder loss, spinal curvature, body-length shortening), cardiotoxicity (decreased heart rate, increased SV-BA distance), hepatotoxicity (reduced liver area index), and neurotoxicity (impaired behavioral ability, and dopamine neuron development deficits), but there was no renal toxicity observed in zebrafish. Additionally, MALDI-MSI analysis showed that BO exposure significantly altered the levels of metabolites, including phospholipids, fatty acids, choline, and amino acids. The contents of PC-34:1, PC-34:2, PI-36:4, PE-36:1, LysoPE-22:5, LysoPC-18:1, FA-18:2, phenylalanine, lysine and glutathione were significantly increased, while the contents of PC-38:6 and PC-40:6 were significantly decreased. Notably, BO elicited a significant alteration in the mRNA expression levels of genes associated with phospholipid metabolism, fatty acid metabolism, choline metabolism, and amino acid metabolism (such as elovl5, chpt1, chka, setd7, hgd). This study revealed that BO exerted toxicity on multiple organs and demonstrated that BO causes metabolic dysregulation in zebrafish. These findings provide a novel insight into the toxicity of BO.