Grape seeds accumulate in huge quantities as byproduct during wine production and are therefore a cheap source for pharmacologically active agents. However, studies prove poor antibacterial activity, and results of analyses are sometimes contradictory. The aim of this study was, thus, to determine the antibacterial activity of grape seed extracts with special focus on the chromatographic characterization of active fractions. In the course of these investigations, extraction protocols were optimized so that microwave-assisted extraction (MAE) guaranteed highest preconcentration efficiency. Proanthocyanidins, monomeric flavonoid aglycones, as well as some of their glycosides could be identified within yielded extracts via high-performance liquid chromatography-mass spectrometry (HPLC-MS). By that means the coherence number of possible isomers of procyanidins was approximated by a newly developed equation. As far as antibacterial activity determined via screening tests is concerned, the extracts generally have been found to be positively responsive toward 10 different gram-positive and gram-negative bacteria strains. After fractionation of the raw extracts, proanthocyanidins P2, P3, P4 and gallate esters P2G and P3G (P = proanthocyanidin consisting of catechin and epicatechin units, n = oligomerization degree, G = gallate ester) were determined as active antibacterial agents toward 10 different pathogens. Only moderate activity was found for monomeric flavonoid fractions.