This study investigates the synthesis of zinc oxide nanoparticles using Penicillium griseofulvum (ZnONPs-PG) and their potential role in preventing DMBA/TPA-induced skin cancer. The synthesis process involved using a 1-mM zinc acetate dihydrate as a precursor in P. griseofulvum. Various analytical techniques, including FTIR spectroscopy, UV-Vis, TEM, XRD, and DLS, were utilized to characterize the ZnONPs. The efficacy of ZnONPs-PG was then evaluated in a DMBA/TPA-induced skin cancer model. Mice were treated topically with DMBA/TPA in acetone (200 μL) over 2 weeks, with treatments continuing for 20 weeks. Results showed 100% tumor occurrence, histological changes, elevated lipid peroxidation (LPO) levels, and decreased antioxidant levels in DMBA/TPA-treated mice. However, topical application of ZnONPs magnificently reverted the tumor occurrence, histological changes, elevated malanoldehyde and hydrogen peroxide levels
decreased antioxidant levels in DMBA/TPA-treated mice. ZnONPs-PG treatment suppressed the increased levels of inflammatory markers (COX-2, iNOS and NF-κB,) and cell proliferation markers (Cyclin-E1, Cyclin D1, VEGF, TGF-β1) exposed mice. In addition, ZnONPs-PG treatment decreased the DMBA/TPA-induced anti-apoptotic Bcl-2 protein and increasing the expression of pro-apoptotic markers (Bax and caspase 3) in skin tissues. Thus, ZnONPs-PG may prevent skin carcinogenesis through its potent antioxidant properties and inhibiting NF-κB-mediated inflammatory and proliferation pathways.