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The challenges in the production of metabolites of medicinal potential from wild plants include low yields, slow growth rates, seasonal variations, genetic variability and regulatory as well as ethical constraints. Overcoming these challenges is of paramount significance and interdisciplinary approaches and innovative strategies are prevalently applied to optimize phytoconstituents’ production, enhance yield, biomass, ensure sustainable consistency and scalability. In this study, we investigated the effects of elicitation with yeast extract and calcium oxide nanoparticles (CaONPs) on in vitro cultures of Swertia chirata (Roxb. ex Fleming) Karsten. Specifically, we examined the effects of different concentrations of CaONPs in combination with different concentrations of yeast extract on various parameters related to callus growth, antioxidant activity, biomass and phytochemical contents. Our results showed that elicitation with yeast extract and CaONPs had significant effects on the growth and characteristics of callus cultures of S. chirata. The treatments involving yeast extract and CaONPs were found to be the most effective in increasing the contents of total flavonoid contents (TFC), total phenolic contents (TPC), amarogentin and mangiferin. These treatments also led to an improvement in the contents of total anthocyanin and alpha tocopherols. Additionally, the DPPH scavenging activity was significantly increased in the treated samples. Furthermore, the treatments involving elicitation with yeast extract and CaONPs also led to significant improvements in callus growth and characteristics. These treatments promoted callus response from an average to an excellent level and improved the color and nature of the callus from yellow to yellow-brown and greenish and from fragile to compact, respectively. The best response was observed in treatments involving 0.20 g/L yeast extract and 90 ug/L CaONPs. Overall, our findings suggest that elicitation with yeast extract and CaONPs can be a useful strategy for promoting the growth, biomass, phytochemical contents and antioxidant activity of callus cultures of S. chirata in comparison to wild plant herbal drug samples.