The aim of the work done was the qualitative and quantitative assessment of a polyphenolic fraction contained in methanolic extracts obtained from the flowering herbs of Marrubium incanum Desr., M. peregrinum L., M. thessalum Boiss. & Heldr., and M. candidissimum L. The samples containing phenolic acids, having undergone acid and alkaline hydrolysis, were investigated by TLC, RP-HPLC, and Q-TOF-MS. In all methanolic extracts of Marrubium species, neochlorogenic, chlorogenic, protocatechuic, p-hydroxybenzoic, caffeic, p-coumaric, gentisic, ferulic acids and flavonoids: rutin, kaempferol rhamnohexoside, kaempferol hexoside, isorhamnetin hexoside, apigenin were identified. This is the first study dedicated to the qualitative and quantitative analysis of polyphenolic compounds in flowering herbs of selected Marrubium sp. Both the total phenolic content (TPC) and the antioxidant activity of water extracts obtained from aerial parts were assessed. TPC was estimated with the spectrophotometric method, whereas the antioxidant activity was evaluated by DPPH assay using Trolox, rutin, caffeic, and gallic acid as reference substances. We also presented the evaluation of antioxidant activity of 70% (v/v) methanolic extracts, that was expressed both as a percentage of DPPH radicals inhibition and IC 50 values (mg/mL). The same Marrubium extracts showed the highest concentration of phenolic acids. Ethanolic (70%, v/v), methanolic, acetone and water extracts of Marrubium sp. were screened for their antibacterial and antifungal activity using the agar microdilution method against a panel of reference strains of 20 microorganisms, including Gram-positive and Gram-negative bacteria and fungi belonging to yeasts.