Nanoparticles (NPs) are defined as particles with a size between one and one hundred nanometers. Recently, NPs have been employed as novel and effective elicitors to increase the yields of chemical compounds in plants. In the present study, zinc acetate and cetyltriammonium bromide (CTAB) were used as reactants to effectively synthesize ZnO-NPs using a straightforward hydrothermal process. Zinc oxide nanorods (ZnO-NPs) were evaluated using an X-ray diffraction pattern (XRD), and transmission electron microscope (TEM). Different Zinc oxide (ZnO) nanorod concentrations (5, 10, 25, 50, and 100 mg/l) were added to the liquid callus medium of Gardenia jasminoides Variegata, and the calli cultures were harvested after 8 days. The amounts of rosmarinic and cinnamic acids were quantified using HPLC. Antioxidant activities were examined by 2,2′- azino - bis (3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS•+) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH). The total content of phenolics and flavonoids has been assessed. It was reported that adding 25 mg/l ZnO nanorods represents the highest fresh weight (6.5 ±0.3 g) compared to the control (5.14± 0.005 g). For accumulation of rosmarinic acid in callus culture of G. jasminoides Variegata using ZnO nanorods, 10 mg/l ZnO nanorods elevated the rosmarinic acid content fivefold compared with the control (250, 50 µg/GFW, respectively), in the same respect 10 mg/l ZnO nanorods recorded the highest antioxidant activity among the other concentrations with both methods ABTS as well as DPPH. The highest cinnamic acid (83 µg/GFW), considered twice its content in the control (39 µg/GFW), was recorded by adding 50 mg/l ZnO nanorods. The antimicrobial activity of the extract of each treatment was also examined against five microbial organisms. The results confirmed a potent activity against candida albicans for treatment No. 3, 5, and 6 extracts. While all the other extracts showed no activity against Bacillus subtilis and Escherichia coli. On the other hand, treatment No. 3 showed moderate activity against Staphylococcus aureus and Shigella flexneri due to the increased production of the active compounds.