Elicitation is one of the effective strategies in plant tissue culture, which stimulates the production of secondary metabolites in plants using specific compounds. This technique can enhance the synthesis of bioactive compounds, such as alkaloids, flavonoids, and terpenes, by stimulating the plant's defense systems. Yeast extract is recognized as a biotic elicitor, rich in vitamins, amino acids, and growth factors. This extract can directly influence the growth and metabolism of the plant, thereby stimulating the plant's defense mechanisms, which in turn increases the production of secondary metabolites. Calendula officinalis, commonly known as marigold, is well-known for its medicinal properties, including anti-inflammatory, antimicrobial, and antioxidant activities. This plant contains bioactive compounds such as flavonoids, carotenoids, and terpenes. The aim of this study was to investigate the effect of yeast extract on callus growth, secondary metabolite production, and antioxidant activity in Calendula officinalis calli. In this study, Calendula officinalis seeds were disinfected and transferred to MS medium. After 20 days, the leaves were transferred to a medium containing 2 mg/L NAA and 1.5 mg/L BAP to induce callus formation. The calli were then cultured in media containing different concentrations of yeast extract (0, 1000, 1200, and 1400 µM). After 4 weeks, various callus characteristics, including fresh and dry weights and callus volume, were measured. Additionally, the phenolic content, flavonoid content, and antioxidant capacity of the calli were measured by extraction and chemical analysis. The results of this study showed that yeast extract significantly increased the production of secondary metabolites in Calendula officinalis calli, and notably enhanced both the antioxidant activity and the growth of the callus. These findings could serve as a foundation for the development of novel methods for the production of pharmaceutical compounds from medicinal plants and the improvement of industrial processes for secondary metabolite production in the pharmaceutical and healthcare industries.