Abstract The aim of this study was to evaluate the phytochemical profile and neuroprotective potential of extracts from underground and aerial parts of two Carlina species: Carlina acaulis (CA) and Carlina vulgaris (CV). Metabolic profiling performed via UHPLC-UV-MS/MS revealed a distinct chemotaxonomic differentiation between the taxa. C. acaulis was defined as a “phenolic-acid” chemotype dominated by 5-O-caffeoylquinic acid (up to 32.11 mg/g in leaves), whereas C. vulgaris was characterized as a “flavonoid” chemotype, distinguished by a unique C-glycoside signature, including carlinoside and schaftoside. A significant aspect of scientific novelty is the first-ever identification of flavonolignans (e.g., salcolin A/B and tricin derivatives) in the roots of both species. In biological assays, the extracts demonstrated a multidirectional neuroprotective mechanism. Good antioxidant properties (DPPH, FRAP assays) were confirmed, particularly in CA leaves and CV inflorescences which revealed 61% of DPPH scavenging, along with the capacity to chelate Fe(II) ions (above 60% for CA inflorescence, CV leaves and CV root), a crucial mechanism in ferroptosis prevention. Furthermore, leaf extracts of both species exhibited effective inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) exceeding 50%, showing kinetic selectivity towards BChE inhibition. These results position Carlina species as a promising source of metabolites supporting the therapy of neurodegenerative diseases. Keywords: Carlina acaulis; Carlina vulgaris; neuroprotection; polyphenols; C-glycosides flavones; flavonolignans; 5-O-caffeoylquinic acid; cholinesterase inhibitors; DPPH; FRAP