The current research involved the synthesis of zinc oxide nanoparticles (ZnO-NPs) using an aqueous extract of Helichrysum cymosum shoots, and subsequent characterization via different analytical methods, such as UV–Vis spectroscopy, Scanning electron microscope (SEM), Energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), Transmission electron microscope (TEM), and zeta potential. The biological effects of the ZnO-NPs were then tested against C3A hepatocyte cells and L6 myocyte cell lines via series of analysis, including cytotoxicity, antioxidant, anti-inflammatory, and antidiabetic effect via enzymatic inhibition. The UV–Vis analysis showed a maximum absorption spectrum at 360, and the TEM analysis reveals a spherical and hexagonal structures, with an average dimension of 28.05–58.3 nm, and the XRD reveals a crystalline hexagonal structure. The zeta potential evaluation indicated that the ZnO-NPs are relatively stable at − 20 mV, and the FTIR analysis identified some important functional group associated with phenolics, carboxylic acid, and amides that are responsible for reducing and stabilizing the ZnO-NPs. The synthesized ZnO-NPs demonstrated cytotoxic effects on the cell lines at higher concentrations (125 µg/mL and 250 µg/mL), complicating the interpretation of the results of the inflammatory and antioxidant assays. However, there was a significant (p < 0.05) increase in the inhibitions of pancreatic lipase, alpha-glucosidase, and alpha-amylase, indicating beneficial antidiabetic effects.