Propolis is a substance manufactured by Apis mellifera and has been widely used in folk medicine due to its high concentration of bioactive compounds. The purpose of the following study was to characterize and evaluate in vitro the antimicrobial properties of propolis on clinical samples and ATCC strains. The chemical characterization of propolis presents a concentration of total polyphenols of 247 +/- 9 mg EAG g-1 MS, flavones and flavonols 75 +/- 4 mg EQ g-1 MS, flavanonones and flavanonols 118 +/- 11 EP g-1 MS. HPLC-DAD identified apigenin, galangin, phenethyl ester of caffeic acid and pinocembrin, in addition to 16 compounds by HPLC MS/MS. Chilean propolis is a natural antimicrobial, showing effectiveness in strains ATCC Staphylococcus aureus, Candida albicans, Trichophyton rubrum and clinical samples of Staphylococcus aureus unlike Escherichia coli. These results demonstrate the antimicrobial effectiveness of the synergy of compounds present in propolis against different human pathogens.