Oregano leaves were extracted using a pilot-scale supercritical fluid extraction plant under a wide range of extraction conditions, with the goal of determining the extraction and fractionation conditions to obtain extracts with optimal antimicrobial activity. In this investigation, the essential oil-rich fractions were selectively precipitated in the second separator, and their chemical composition and antimicrobial activity were investigated. Gas chromatography-mass spectrometry analysis of the various fractions resulted in the identification of 27 compounds of the essential oil. The main components of these fractions were carvacrol, trans-sabinene hydrate, cis-piperitol, borneol, terpinen-4-ol, and linalool. Antimicrobial activity was investigated by the disk diffusion and broth dilution methods against six different microbial species, including two gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), two gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), a yeast (Candida albicans), and a fungus (Aspergillus niger). All of the supercritical fluid extraction fractions obtained showed antimicrobial activity against all of the microorganisms tested, although the most active fraction was the one obtained in experiment 5 (fraction was obtained with 7% ethanol at 150 bar and 40 degrees C). C. albicans was the most sensitive microorganism to the oregano extracts, whereas the least susceptible was A. niger. Carvacrol, sabinene hydrate, borneol, and linalool standards also showed antimicrobial activity against all of the microorganisms tested, with carvacrol being the most effective. Consequently, it was confirmed that essential oil from experiment 5, with the best antimicrobial activity, also presented the highest quantity of carvacrol.