Ballota macrodonta Boiss. et Balansa., is a traditionally used endemic medicinal plant used to manage a plethora of diseases in Turkey. Nonetheless, few studies have endeavoured to highlight its therapeutic potential and its phytochemical profile. We hypothesized that B. macrodonta would possess multi-pharmacological propensities and bioactive compounds that would justify its use as a folk remedy. Aerial part of B. macrodonta was assessed for its antioxidant, antimicrobial, and enzymatic inhibitory potential using different extraction solvents. The total bioactive components determination and compound identification using High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) method were used to correlate the biological properties. HPLC-DAD profile showed the presence of 19 components (e.g. hydroxybenzoic acid, catechin hydrate, vanillic acid, caffeic acid, syringic acid, epicatechin, coumaric acid, ferulic acid, luteolin-7-O-glucoside, rutin, and quercetin). The water and methanolic extracts showed high total phenolic and flavonoid contents, respectively. The overall antioxidant capacity of the water extract was higher than the one of the methanol extract. The extracts showed inhibitory activity against cholinesterases, tyrosinase, α-amylase and α-glucosidase. The extracts also presented antimicrobial activity against eight Gram positive/negative bacteria (MIC – 0.05–0.15 mg/mL) and eight fungi (MIC – 0.0125–0.20 mg/mL). The presence of active biomolecules with multi-pharmacological properties makes B. macrodonta a potential source of health-promoting compounds that can be exploited as a novel medicinal herbal product.