CuO engineered nanoparticles (ENPs) are widely used across various industries, resulting in their environmental release and subsequent transformation. This study examined the impact of chemical (sulphidation), biological (bovine serum albumin (BSA) corona), and double transformations (sulphidation + BSA coating) of CuO ENPs on the immobilisation of Daphnia magna (0.63–10 mg Cu/L) and the inhibition of root growth of Lepidium sativum (10–160 mg Cu/L). Transformations of CuO ENPs altered their chemical composition, morphology, and surface chemistry; the extent of changes depended on the transformation type. Dual transformations of CuO ENPs generated properties distinct from pristine and singly transformed ENPs. The transformations affected CuO ENP behaviour in aqueous media, including aggregation, dissolution rate, and ζ potential, ultimately influencing toxicity. Sulphidation increased CuO ENP dissolution fivefold, resulting in complete D. magna immobilisation. In contrast, BSA coating mitigated toxicity across all ENPs, enhancing daphnia mobility by 30–95 %. High CuO ENP concentrations inhibited L. sativum root growth, while all transformed ENPs exhibited reduced phytotoxicity. The reduced metal ion release from CuO ENPs only partly explained the lower toxicity of transformed CuO ENPs. Interactions between ENPs and biota were additionally modulated by ζ potential, aggregation kinetics, and organic surface coatings that restrict direct contact, and inorganic compounds. These results underscore the critical role of ENP transformations in modulating their toxicity to organisms.