Propolis is a bee product known to humans for over two thousand years. Its antimicrobial properties have been the subject of numerous studies, with alcohol-based (ethanol) propolis extracts being the most commonly used. Although effective and frequently utilized, such extracts are not suitable for some conditions and patients in human and veterinary medicine due to the effects of ethanol. Motivated by these facts, our research employed an alcohol-free, standardized and harmless propolis extract. We aimed to determine its antimicrobial effectiveness against the bacterium S. aureus and the fungus C. albicans, as well as to explore the mechanisms of its antibacterial action. We determined the concentrations of active substances in the extract using the HPLC method and then examined its effects on the test microorganisms through a series of microbiological procedures. In addition to flavonoids, the alcohol-free propolis extract contained ferulic acid, p-coumaric acid, caffeic acid, and CAPE. The minimum inhibitory concentration (MIC) of propolis against S. aureus was 0.883 % and 1.324 % propolis, while against C. albicans, it was 0.883 % propolis. By measuring the concentration of proteins and double-stranded DNA (dsDNA) in the extracellular matrix after applying propolis to S. aureus, we confirmed that changes occurred in the bacterial membrane and cell wall, leading to the leakage of intracellular material into the extracellular matrix. Furthermore, by employing a time-kill assay, we concluded that propolis has a bactericidal effect on S. aureus and a fungistatic effect on C. albicans. In conclusion, our research has demonstrated that the standardized and alcohol-free propolis extract disrupts the integrity of the bacterial membrane and cell wall, causing the release of cellular material (proteins and double-stranded DNA) from S. aureus. Additionally, we have proven the fungistatic activity of the extract against the fungus C. albicans, thus confirming the full antimicrobial potential of the alcohol-free propolis extract. The obtained results can serve as a valuable basis for further research and potential commercial applications.