Essential oils are natural products that have great antimicrobial potential value against many fungi and bacteria. Rosa damascena Mill. is one of the most important aromatic species of the Rosaceae family from which essential oil and economically valuable products can be obtained. The present study was designed to investigate the major compositions of the essential oil of this plant in Isfahan region of Iran and to identify its antibacterial and antifungal effects against 11 microorganisms causing human diseases and food spoilage. The essential oil was extracted by using the Clevenger apparatus and was analyzed by gas chromatography-mass spectrometry (GC-MS) technique. Its antimicrobial activity was evaluated by well diffusion, minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC). The results showed that the most important compounds of the essential oil were nonadecane (24.72%), heneicosane (19.325%), oleic acid (17.63%), and citronellol (12.61%). The results also showed that the highest inhibition zone of rose essential oil was against Aspergillus brasiliensis (15.00 ± 0.00 mm) and had a significant effect on Klebsiella pneumoniae (~ 8.00 mm). Also the rose oil had a significant inhibition and lethal effect against Candida albicans (MIC and MBC ~ 125 μg/mL), which is equivalent to the nystatin antibiotic (~ 125 μg/mL). Therefore, the essential oil of Damask rose can be considered as an alternative natural product for the prevention and treatment of fungal diseases in humans and against food spoilage as well.