The chemical composition and antimicrobial activity of essential oil-rich fractions obtained by supercritical CO2 extraction from Rosmarinus officinalis L. were investigated. Gas chromatography-mass spectroscopy analysis of these fractions resulted in the identification of 33 compounds of the essential oil. The main components of these fractions were alpha-pinene, 1,8-cineole, camphor, verbenone, and borneol, constituting ca. 80% of the total oil. The antimicrobial activity was investigated by the disc diffusion and broth dilution methods against six microbial species, including gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), a yeast (Candida albicans), and a fungus (Aspergillus niger). All of the essential oil-rich fractions obtained showed antimicrobial activity against all of the microorganisms tested, with inhibition zones and minimal bactericidal and fungicidal concentration values in the range of 17 to 33 mm and 2.25 to 0.25 mg/ml, respectively. The most active fraction was the one obtained in experiment 4 (4% ethanol as modifier; extraction pressure, 25 MPa; extraction temperature, 60 degrees C). S. aureus was found to be the most sensitive bacteria to the rosemary extracts, whereas the least susceptible was A. niger. alpha-Pinene, 1,8-cineole, camphor, verbenone, and borneol standards also showed antimicrobial activity against all the microorganisms tested, borneol being the most effective followed by camphor and verbenone. In that way, it was confirmed that essential oil from experiment 4, with the best antimicrobial activity, presented the highest quantity of camphor, borneol, and verbenone.