The chemical composition of commercial thyme oils, freshly hydrodistilled EO (essetntial oil) from dried thyme herb and thymol, the main thyme oil constituent, were analyzed in the aspect of possible cytotoxic effect against MCF-7 breast cancer and normal L929 mouse fibroblast cell lines. Based on the GC-MS analysis, it was found that the commercial essential oils revealed similarities in their chemical composition. The content of main components such as thymol, linalool and α-pinene was almost equal. Interestingly, the EO obtained by hydrodistillation from Thymi herba showed considerable differences in the percentage content of some main constituents. The reason for the differences may be caused by the intraspecific chemical variability of T. vulgaris L. Four types of tested EOs can be classified as a ‘thymol’ chemotype, with thymol as the predominant compound. The thymol alone and the freshly hydrodistilled EO demonstrated the highest cytotoxic effect against used cell lines. The difference in IC50 values suggests more sensitive L929 cells are more sensitive in both the CCK-8 assay (except EOs Kawon) and the NRU assay.