The essential oil of the resin of Pistacia lentiscus var. Chia (mastic oil) was studied in vitro against a wide range of foodborne pathogenic and spoilage microorganisms with a diffusion and a dilution method. Furthermore its chemical composition was analyzed by means of Gas Chromatography-Mass Spectrometry (GC-MS) and the possibility of using the essential oil in food preservation was discussed. The Minimal Inhibitory Concentrations (MICs) of mastic oil were estimated for 6 species of bacteria (Bacillus cereus, Campylobacter jejuni, Clostridium perfringens, Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus), 2 species of yeasts (Saccharomyces cerevisiae and Zygosaccharomyces bailii) and 3 species of fungi (Penicillium roquefortii, Aspergillus flavus and Eurotium amstelodami). GC-MS analysis revealed a chemotype dominated by monoterpenes, principally α-pinene and β-myrcene comprising more than 90 % of the mastic oil. Both methods showed Cl. perfringens as the most susceptible microorganism followed by S. cerevisiae and Z. bailii. With the exception of C. jejuni, Gram-positive were found to be more susceptible to the essential oil than Gram-negative microorganisms and all fungi appeared very resistant to mastic oil. Based on the observed MICs, the contribution of mastic oil to the preservation of bakery/confectionary products at the amounts currently used for flavoring purposes is likely to be negligible.