Display omitted •Tanacetum macrophyllum and Telekia speciosa phytochemical profilings were analyzed.•Acylquinic acids, methoxylated flavonoids and sesquiterpene lactones were annotated.•Chemometric and multivariate analyses of biological activity were performed.•T. speciosa has prominent cholinesterase and tyrosinase inhibitory potential.•T. macrophyllum shows the highest radical scavenging activity and reducing power. Asteraceae species Tanacetum macrophyllum (Walds. & Kit.) Sch.Bip. (Tm) and Telekia speciosa (Schreb.) Baumg. (Ts) are renowned for their ethnomedicinal use. This work aims to provide an in-depth characterization of the methanol-aqueous extracts from the flower heads, aerial parts and roots of the species. Annotation and dereplication of 113 secondary metabolites was achieved in the studied samples; 63 compounds are reported in the species for the first time. Herein, 38 acylquinic acids analogues, 31 flavonols and flavones, 17 sesquiterpene lactones together with phenylethanoid glycosides and sugar esters were evidenced in the assayed extracts. The principal component and heat-map analysis identified 20 marker metabolites that revealed herbal drug-specific chemical patterns: phenylethanoid glycosides, quinic, chlorogenic and 3-feruloyl-4-caffeoylquinic acid, patuletin, eupatorin, quercetagetin-trimethyl ether, artecalin and artemisiifolin had the highest contribution in variance. The esters of caffeic/ferulic acid-hexoside were exclusively produced by T. speciosa while methoxylated flavonoids were evidenced in T. macrophyllum. According to the multivariate analysis of biological potential, radical scavenging activity and reducing power accounted mostly for the stronger antioxidant capacity of Tm in comparison with Ts. Telekia specosa aerial parts and roots had marked impact on acetyl- and butyrylcholinesterase (up to 4.82 ± 0.22 mg galantamine equivalent (GALAE)/g and 3.20 ± 0.30 mg GALAE/g, respectively) and tyrosinase (up to 119.00 ± 0.97 mg kojic acid equivalent/g) inhibition. Tanacetum macrophyllum aerial parts and flower heads were beneficial for attenuating carbohydrate metabolism disorders witnessed by α-glucosidase (1.45 ± 0.27 mmol acarbose equivalent (ACAE)/g) and α-amylase (0.65 ± 0.04 mmol ACAE/g) inhibitory activity. Tanacetum macrophyllum flower heads and T. speciosa roots can be recommended for production of raw material with antioxidant and enzyme inhibitory potential.