Essential oils (EOs) are known to inhibit the growth of a wide range of microorganisms. Particularly interesting is the possible use of EOs to treat multidrug-resistant cystic fibrosis (CF) pathogens. We tested the essential oil (EO) from Origanum vulgare for in vitro antimicrobial activity, against three of the major human opportunistic pathogens responsible for respiratory infections in CF patients; these are methicillin-resistant Staphylococcus aureus, Stenotrophomonas maltophilia and Achromobacter xylosoxidans. Antibiotic susceptibility of each strain was previously tested by the standard disk diffusion method. Most strains were resistant to multiple antibiotics and could be defined as multi-drug-resistant (MDR). The antibacterial activity of O. vulgare EO (OEO) against a panel of 59 bacterial strains was evaluated, with MIC and MBC determined at 24, 48 and 72 hours by a microdilution method. The OEO was effective against all tested strains, although to a different extent. The MBC and MIC of OEO for S. aureus strains were either lower or equal to 0.50%, v/v, for A. xylosoxidans strains were lower or equal to 1% and 0.50%, v/v, respectively; and for S. maltophilia strains were lower or equal to 0.25%, v/v. The results from this study suggest that OEO might exert a role as an antimicrobial in the treatment of CF infections.