ZnO (nano)structures remain of great interest in biomedical applications due to their unique properties and possible morphologies. Biocompatibility of typically fabricated ZnO structures remains questionable and they still lack desired biological functions, whence their functionalization is of high interest. In this work, we fabricated micro-sized ZnO hierarchical flower-like structures using facile template-free hydrothermal method to act as carriers for the delivery of gold nanoparticles (Au) and/or Biotin (Vitamin B) to cells. Au nanoparticles (~24 nm), as well as Biotin molecules were successfully deposited on the ZnO surface due to non-covalent physical interactions. We have then cultured two cells lines: SH-SY5Y (human malignant neural) and HEK-293 (human non-malignant) and observed that ZnO hierarchical particles exhibited cell line-dependent cytotoxicity. It appeared that further functionalization of ZnO with Au nanoparticles and subsequently with Biotin led to lower discrepancy between the two cell lines response indicating that cytotoxic pathways of pure ZnO were masked by the available surface adsorbed particles (Au/Biotin). Two-photon immunocytochemistry microscopy further confirmed that Biotin decorated particles affected neuroblastoma cells cytoskeleton. These findings contribute to the understanding of cytotoxic pathways of surface-decorated nano-micro-structures made from ZnO with two molecules typically used in anticancer and regenerative medicine therapies.