Honey, the primary product of honey bees, has been used as a medicine for thousands of years, but antimicrobial effects of honey still are not fully understood. For this study, 14 samples of honey were collected from different locations of southern Dalmatia and Croatian mainland. The main goal was determining the minimum inhibitory concentration of strawberry tree, heather, mandarin and chestnut honey. Used pathogens were bacteria E. coli, S. aureus, H. pylori, and M. fortuitum, and the fungus C. albicans. We used method of plating the bacteria on a microtitre plate, in which are serial dilutions of honey previously added. After incubation, the results are read with a spectrophotometer (540 nm), and was determined the minimum concentration of honey that inhibits growth of more than 50%, or 80% of bacteria. Best antimicrobial effects show strawberry tree and heather honey, while the weakest mandarin honey. By modified Folin-Ciocalteu method was determined the amount of total phenols, the amount of flavonoids was determined colorimetric with AlCl3, and antioxidant activity of honey was determined by FRAP method and the method of DPPH radicals. The lowest concentration of total phenols, flavonoids and antioxidant activity shows strawberry tree honey, and the highest amount of flavonoids has mandarin honey. The highest values of total phenols and antioxidant activity has heather honey. The antimicrobial effectiveness depends primarily on botanical and, subsequently, geographical origin.