In this study, antimicrobial effects of two accession of Thymus fedtschenkoi originated from Semnan and Zanjan against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Bacillus subtilis were investigated. The essential oil was extracted by Clevenger apparatus and compounds identified by GC and GC-MS instruments. Disc diffusion and microdilution methods were used to determine antimicrobial activity. The experiment was done on factorial design based on complete randomized design. The effect of microorganism type (as first factor) and essential oil concentration and antibiotics (as second factor) and their interaction was evaluated. According to the results, essential oil yield was %0.73 and %0.95 in Zanjan and %0.83 and %0.86 in Semnan accession in two successive years. The main chemical compounds of Zanjan accession was p-cymene, linalool, thymol and carvacrol and Semnan accession was p-cymene, thymol and carvacrol. Statistical analysis showed that the effect of microorganism type, essential oil concentration and their interaction on inhibition zone was significant at 1% level. In Semnan accession, the highest diameter of inhibition zone was observed in C. albicans and in dilution of one fifth of essential oil. C. albicans and B. subtilis had the most sensivity to Zanjan accession and C. albicans to Semnan accession. The observed difference in antimicrobial effects can be related to difference in chemical composition and their activity.