The polyphenolic composition and anti-inflammatory activity of selected Thymus species ( T. longicaulis C. Presl, T. praecox Opiz subsp. polytrichus (A. Kerner Ex Borbás) Jalas, T. pulegioides L., T. serpyllum L. subsp. serpyllum and T. striatus Vahl) was studied in comparison with T. vulgaris , as an officinal medicinal plant, to evaluate the biomedical potential of these Croatian wild-growing species. This paper provides a first insight into the polyphenolic profiles of T . praecox subsp. polytrichus and T. striatus . Also, some flavone and hydroxycinnamic derivatives were at first identified in the other studied species by LC-DAD-ESI–MS/MS analysis. Rosmarinic acid and glycosides of scutellarein and luteolin were found to be the most abundant polyphenolic constituents. The Thymus extracts inhibited Src tyrosine kinase activity and they reduced the production of proinflammatory cytokine interleukin-6 in Balb/C mice splenocytes. The obtained results highlighted T. longicaulis as a potential source of herbal drugs with anti-inflammatory properties and/or novel functional food rich in flavonoids and phenolic acids.