Medicinal plants are considered to be a major source of biologically active compounds, which provides unlimited opportunities for their use either as medical treatments or as novel drug formulations. The focus of our study was on basic phytochemical analysis and in vitro examination of the biological activity of Alchemilla vulgaris L. Methanolic extracts of above ground parts and roots of A. vulgaris (AVA and AVR, respectively) were prepared by maceration for 72h. Phytochemical profile of extracts was evaluated by spectrophotometric determinations of phenolic compounds and HPLC-PDA analysis. AVA and AVR were analysed for their antioxidant efficacy as total antioxidant capacity, metal chelation and reducing power ability, inhibition of lipid peroxidation as well as their potential to neutralise DPPH, ABTS, and OH radicals. Microdilution method was employed to investigate the antibacterial and antifungal activity of extracts against nine ATCC and isolates of bacteria and ten fungal strains from biological samples. Anti-inflammatory activity of the extracts was evaluated using cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) assays and the assay for determination of COX-2 gene expression, while biocompatibility of extracts was assessed by MTT assay. Our results revealed the high amount of phenolic compounds in both extracts; especially they were rich in condensed tannins. Ellagic acid and catechin were tentatively identified in AVA and AVR, respectively. Full biocompatibility as well as remarkable bioactivity were observed for both extracts in all employed assays, so our further investigations will be focused on the identification of active constituents in A. vulgaris and the molecular mechanisms of their action. •A. vulgaris aerial parts and roots extracts were analysed.•Methanol extracts of aerial parts and roots were rich in phenolic compounds.•Ellagic acid was detected in aboveground parts and (+)-catechin in the roots.•Extracts showed remarkable antioxidant and anti-inflammatory activities.•Aerial parts extract exhibited better activity in all employed assays.