Increasing antimicrobial resistance has caused a great interest in natural products as alternatives or potentiators of antibiotics. The objective of this study was to isolate individual tannins from crude chestnut extract as well as to determine the influence of both crude extracts (tannic acid extract, chestnut extract) and individual pure tannins (gallic acid, vescalin, vescalagin, castalin, castalagin) on the growth of Gram-positiveStaphylococcus aureusbacteria. Their antibacterial activity was monitored by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as well as the duration of the lag phase, growth rate and generation time. The effect of growth medium strength on the MIC of different tannins was also investigated. Bacterial growth was followed spectrophotometrically, and MIC values were determined by the microdilution method. The MIC values of various isolated compounds allowed us to determine the bioactive compounds and their contribution to antimicrobial activity. It was found that MIC values increase with increasing growth medium strength and that the lag phase lengthens with increasing tannin concentrations, while the growth rates decrease. Comparing the results of the two studies, the antimicrobial activity of tannins againstS. aureuswas not as pronounced as in the case ofE. coli, which may indicate that a different mechanism of action is responsible for the antimicrobial effects of tannins on Gram-positive than on Gram-negative bacteria, or that a different mechanism is more pronounced.