Withania somnifera is an important medicinal plant due to the presence of secondary metabolites. Nanoparticles (NPs) have elicitor activity for the enhancement of secondary metabolites biosynthesis in plants. W. somnifera plants were grown in-vitro and in-vivo and treated with homologous series of Zn-Ag NPs, Ni, and CdSe. Four NPs having different molar ratio of Zn and Ag have been used for the treatment. NP1, NP2, NP3, and NP4 Exhibit 19:1, 9:1, 3:1 and 1:1 molar ratios between Zn and Ag. Among all the treated NPs NP1 showed maximum enhancement in photosynthesis rate, transpiration rate, withanolide content and increase in some carbohydrates. Genes involved in photosynthesis, Calvin cycle, carbohydrate metabolism, and withanolide biosynthesis showed up regulation in NP1 and NP3 treatments. Zn-Ag NPs showed the photocatalytic and elicitor activity which helped in the light absorption and increased oxidative stress. NP2, NP4, Ni, and CdSe treatments showed the negative impact on withanolide biosynthesis and withanolide content. NP2, NP4, Ni, CdSe treated samples showed down regulation of most of the carbohydrate and withanolide pathway genes and lower withanolide content in comparison with water treated plants. CdSe caused excess cell damage, therefore, decrease in withanolide content was recorded. Gas exchange and electron transfer rate increased in CdSe and NP1 increases due to their semiconducting properties. CdSe and NP1 can liberate significant number of electrons under the light exposure, which behaves like carriers. Cyclic electron flux was observed in all the treatments but lowest in control condition. Cyclic electron flux not only manages the electron flow around PSI under stress but also considered as essential for photosynthesis and plant growth under control condition.