The aim of the research was to explore qualitative and quantitative phytochemical constituents of different extracts from Agrimonia eupatoria L., Hypericum alpestre subsp. polygonifolium (Rupr.) Avet. & Takht., Rumex obtusifolius Willd and Sanguisorba officinalis L. and analyze their bioactive constituents using TLC-bioautography technique. In TLC analysis different solvent systems as mobile phases were used in order to make assumptions about the nature of active antibacterial compounds. The highest total phenolic, flavonoid and tannin content was detected in methanol extract of A. eupatoria (358.9 µg Gallic acid equivalent per mg dry weight), acetone extract of H. alpestre (216.75 µg Quercetin equivalent per mg dry weight) and acetone extract of R. obtusifolius (76.24 µg Catechin equivalent per mg dry weight), respectively. TLC-bioautography analysis using mobile phases with different polarity allowed to separate different bands from tested plants methanol and acetone extracts possessing antibacterial activity against Staphylococcus aureus MDC 5233. Thus, it was revealed, that polar compounds could have considerable contribution to the antibacterial activity of acetone and methanol extracts of H. alpestre and R. obtusifolius. It was hypothesized, that complex compounds can be responsible for the antibacterial action of extracts of A. eupatoria and S. officinalis. Based on obtained data, A. eupatoria, H. alpestre, R. obtusifolius and S. officinalis plants can be recommended as sources of new antibacterial agents.