Bacteria, algae, and fungi are the organisms affected by biocidal substances, which are widely used. Apart from these, they can also have toxic effects on various aquatic organisms. Copper pyrithione (CPT) and zinc pyrithione (ZPT) are used as an alternative to tributyltin, which is forbidden and known to be highly toxic to the aquatic ecosystem. However, there is lacking information about histopathological alterations and endocrine disrupting effects of these substances. Therefore, this study was aimed to investigate the effects of ZPT, CPT, and their mixtures on the histological changes in the tissues and on the vitellogenin hormone in male zebrafish. Substances caused hyperemia, epithelial lifting, telangiectasis, and hyperplasia in the gill tissue. In the liver tissue, hyperemia, hydropic degeneration and necrosis were observed. In addition, apoptosis was found in the gill, liver, and testicle of the tissues as a result of the TUNEL assay. Vitellogenin hormone, on the other hand, increased in the experimental groups compared to the control groups (p < 0.05). According to the obtained results, these substances, which are used as alternative antifouling materials, show as endocrine disrupting compounds by acting on vitellogenin as well as causing histopathological changes.