In this paper, an attempt was made to carry out a chromatographic analysis of fractions of extracts containing substances with bactericidal activity, obtained from selected hop varieties. By searching for the ideal mobile phase, thin-layer chromatographic (TLC) analysis of all selected hop varieties was performed by use of silica/dichloromethane isopropanol systems. Using direct bioautography as a detection method, extracts with the best bactericidal properties were selected. Specific fractions were isolated using preparative planar chromatography. Depending on the location of the fraction, a suitable mobile phase was applied to ensure the best separation of the tested extracts. The isolated fractions were subjected to TLC analysis and direct bioautography using the Bacillus subtilis strain. The analyzed extracts of selected hop varieties represent promising material for work in the search for new substances with bactericidal/bacteriostatic activity.