Zinc is an essential element that is readily available, inexpensive, and ecologically friendly, and it will be employed in both theranostic (diagnostic and therapeutic) applications in current industrial developments. Our current work used the green chemical approach to illuminate the zinc oxide nanoparticles (ZnO NPs) synthesized from Couroupita guianensis (CG) flower extract. Ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), Field Emission Scanning Electron Microscopy (FE-SEM), Energy-dispersive X-ray (EDX), X-ray diffraction (XRD) embedded and techniques characterize the produced CG-ZnO NPs and determine their characteristics. Following the addition of CG-ZnO NPs, the antioxidant, antibacterial, and human sperm viability followed by DNA damage inhibition may be assessed using the comet test. The characterization results show that phenols, phytosterols, aromatic nitrides, and phytosterols from the FT-IR functional groups, UV-Vis of 340 nm as ZnO NPs confirmatory, followed by XRD with a 2-theta value of 101o shows the sample’s crystallinity and its pure phase were validated by the XRD signal and FE-SEM with 14–18 nm in size and a spherical structure, then EDX with Zn particles confirm the presence of Zn. According to the results, CG-ZnO NPs form zones with diameters of 18 mm, 15 mm, 13 mm, 11 mm, and 7 mm against both Gram-positive and Gram-negative bacteria. In addition, findings from antioxidant scavenging of free radicals quenching against specified DPPH methanol reveal that CG-ZnO NPs with 100 µg/ml (71.32%) are enough to act against free radicals, approximately equal to control ascorbic acid (72%). As a first effort, CG-ZnO NPs prevent DNA damage in human sperm cells against H2O2 and UV radiation compared to the negative control. CG-ZnO NPs improved sperm cell viability and inhibited DNA damage when compared to control and H₂O₂. This research evaluates the outcome of the Comet Assay Software Project (CASP) for evaluating comet photographs. The automated CASP system aids in the assessment of sperm motility and quality. Comet formation in sperm cells, indicating DNA damage, was observed, and sperm cells treated with CG-ZnO NPs were recognized and validated using CASP version 1.2.2. As a consequence, our CG-ZnO NPs may be used as cryoprotectants against the freeze/thaw of artificial reproduction technology centres, protecting them from ROS free radicals for extended periods. Overall, the comparison results are consistent in both antioxidants and antibacterials, with notable results in sperm DNA damage inhibition having the potential for usage in various therapeutic domains. © 2025 Elsevier B.V., All rights reserved.