Gymnema sylvestre is a plant that is enriched in bioactive compounds. In particular, gymnemic acids (GA) and phenolic compounds (PC) are pharmaceutically important. There is a commercial demand for naturally occurring bioactive compounds, but their availability is limited due to geographical and seasonal variations. The elicitation approach can enhance the biosynthesis of phytochemicals during in vitro culture of G. sylvestre. Here, to further improve gymnemic acid II (GA II) and phenolic compounds (PC) production by G. sylvestre, cell suspension cultures (CSC), which has attracted attention for the production of essential phytochemicals, was explored using copper oxide nanoparticles (CuO NPs). Callus was obtained on MS medium containing 2,4-dichlorophenoxyacetic acid, kinetin, phytoagar, and sucrose. Agar-free MS medium was used to initiate CSC, which was treated with three concentrations of CuO NPs (1, 3 or 5 mg/L). Treatment for 48 h with 3 mg/L CuO NPs resulted in the greatest yields of GA II, total phenolics, and flavonoids. The cultures also displayed pronounced antioxidant, antidiabetic, anti-inflammatory, antibacterial, antifungal, and anticancer activities. The use of CuO NPs (3 mg/L) significantly increased the production of GA II (nine-fold) and PC compared to unamended CSC. We propose that CSC and use of nanoparticles (NPs) as a new generation of elicitors, offer a suitable prospect for the production of bioactive compounds.