The volatile oils obtained from the leaves and flowers of the Croatian endemic plant Centaurea ragusina L. were evaluated for antimicrobial activity. The chemical compositions, determined by GC and GC-MS, were complex, with sesquiterpenes the most abundant class of compound. Nonterpene hydrocarbons were also identified in high quantities, while acids and ketones were found in high quantities only in the leaf volatile oil. Aldehydes, alcohols, esters and other compounds were identified in lower quantities. Antimicrobial effects of the volatile samples were investigated by using the disc diffusion and microdilution assays. They were found to inhibit a wide range of bacteria and fungi, causing in vitro growth inhibition at a concentration of 250 microg/mL. The volatile oils displayed great antibacterial potential with MIC values ranging from 16 to 128 microg/mL against the Gram-positive bacteria Bacillus cereus, Clostridium perfringens, Enterococcus faecalis and Staphylococcus aureus, and lesser activity against the Gram-negative bacteria Cronobacter sakazakii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as well as fungal strains (MIC = 32-128 microg/mL).