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Thymus species have found remarkable importance in food and medicine industries. The present study investigates the potential effect of Ag nanoparticle elicitors on proliferation of callus, and production of carvacrol and thymol in Zataria multiflora and three Thymus species. Firstly, callus was induced on Murashige and Skoog (MS) medium containing 2 mg l−1 of 2, 4-dichlorophenoxy acetic acid (2,4-D) and 1 mg l−1 of kinetin (Kin)). Secondly, the effects of two different concentrations of Ag nanoparticles (4 and 8 mg l-1) were studied on callus growth and its secondary metabolites production. Results elucidated that after elicitation by 8 mg l-1 ofAg NPs, significantly the highest callus growth rate (CGR) (0.02 mm day-1), callus fresh mass (CFM) (0.99 g), and carvacrol (0.68 mg l-1) and thymol (11.09 mg l-1) content was achieved. Comparing different Thymus species, notably the greatest carvacrol and thymol amount was obtained in .kotschyanus Boiss. & Hohen. and T. Daenesis Čelak. at 8 mg l-1 concentration ofAg NPs. Hence, it is evident that the stimulation by NPs is dose-dependent. This study has potential to be commercially applied for the enhancement of pharmaceutical compounds in different species of Thymus.