Secondary metabolites play an important role in plant responses to environmental stresses and some of these compounds are significantly increased under stress conditions. The use of biotic and abiotic elicitors for promoting the production of secondary metabolites reduces the processing time to achieve high concentrations of the metabolite. In this study, the effects of pathogenic fungus, Rhizoctonia solani (AG2-2) SBUka2, as biotic elicitor, were studied to better understanding of plant reactions to microorganisms and enhance yield and quality of secondary metabolites of Thymus daenensis in tissue culture. In this way, rosmarinic acid and caffeic acid contents of two ecotypes (Ilam and Isfahan) of T. daenensis were determined by High Performance Thin Layer Chromatogharapghy (HPTLC), while essential oil variations were studied by Gas Chromatoghraphy-Mass Spectrometry (GC-MS). The results showed that in the presence of R. solani, rosmarinic acid content of inoculated plant was decreased by 26.82% and 8.25% in Ilam and Isfahan ecotypes, respectively whereas caffeic acid content decreased to 4.51 and 4.16, respectively. The analysis of essential oils exhibited that in the presence of R. solani, the yield of essential oils increased by 33% and 20% in Isfahan and Ilam types, respectively. In addition, the content of some essential oil compositions such as thymol and carvacrol were increased in infected plants whereas other compounds such as para-cymene and gama-terpinene were decreased.