Pomegranate trees are tropical and subtropical shrubs with nutritional benefits and pharmaceutical and therapeutic uses. Antioxidative systems protect the structure and function of cellular membranes. This study demonstrated the connection between oxidative stress generated by excess nanoparticles ZnO (ZnO-NPs) accumulation in pomegranate calli and the involvement of thiol groups and volatile and semi-volatile compounds in alleviating this stress. The effect of the non-enzymatic antioxidant system was studied using callus treated with three levels of ZnO-NPs or bulk particles (ZnO-BPs). With rising ZnO levels in the media, callus growth was gradually decreased by ZnO in both forms (NPs and BPs). Malondialdehyde (MDA) measurements revealed that different concentrations of both forms promoted lipid peroxidation. The supply of both forms had a considerable stimulatory influence on the cysteine (Cys) content in calli. Raised ZnO-NP concentrations increased glutathione (GSH) and non-protein thiols (NPTs) content in calli, but higher ZnO-BP concentrations lowered their content. Conversely, ZnO-NP levels reduced the protein thiols (PTs) content in calli, but ZnO-BP concentrations increased their content. GC-MS analysis was employed to investigate the volatile and semi-volatile chemical profiles within calli following exposure to 0 and 150 μg mL⁻¹ of ZnO in both forms. GC-MS analysis detected 77, 67, and 83 compounds in ZnO-treated calli, of which 14, 16, and 20 with a similarity value greater than 70%, based on a NIST library, were recognized as metabolites for ZnO untreated and NPs- and BPs-treated calli, respectively. Six substances, including five alkanes and one morphinan, showed similarities in metabolite composition between control and NPs- or BPs-treated calli. ZnO-NPs-treated calli contained two alkane compounds only similar to the control, but ZnO-BPs-treated calli had six metabolites, including four alkanes, one carboxylic acid, and one ester. However, eight alkanes were similar within the callus treated with NPs and BPs.