Chromochloris zofingiensis is a promising microalga for producing high-value metabolites, particularly astaxanthin and lipids. This study evaluated the concentration-dependent effects of zinc oxide nanoparticles on growth, biomass production, photosynthetic pigments, astaxanthin, protein, and lipid accumulation in C. zofingiensis. The commercial ZnO-NPs, characterized before biological application, exhibited crystalline wurtzite structures with nanosized primary particles of approximately 50–100 nm that formed larger agglomerates. The microalga was cultivated in a standard BG-11 control medium containing zinc sulfate heptahydrate (0.22 mg L⁻¹) and in zinc-free BG-11 medium supplemented with ZnO-NPs at concentrations of 0, 0.06, 6, 30, and 60 mg L⁻¹. Physiological and biochemical responses were assessed through growth analysis, biomass measurement, pigment and astaxanthin determination, and proximate biochemical analysis. ZnO-NPs markedly altered microalgal metabolism in a dose-dependent manner. Growth and biomass were relatively maintained at low to moderate concentrations but declined under higher exposure levels. Chlorophyll a+b showed strong sensitivity to ZnO-NPs, with substantial reductions at 6–60 mg L⁻¹, while total carotenoids were comparatively more retained. Interestingly, the highest astaxanthin concentration was obtained at 6 mg L⁻¹, indicating that moderate ZnO-NP exposure induced a protective secondary carotenoid response. Protein content increased with mild exposure, whereas lipid content decreased at 6 mg L⁻¹, suggesting a redistribution of carbon flux toward stress-defense metabolism rather than storage lipid accumulation. These findings reveal a physiological boundary between ZnO-NP toxicity and nano-biostimulation in C. zofingiensis. Controlled exposure to ZnO-NPs may therefore provide a promising strategy for enhancing astaxanthin production in microalgal biotechnology.