Objective: To determine the phytochemical characterization and antibacterial activity of Melissa officinalis essential oil against bacteria responsible for nosocomial infections. Methods: The phytochemical characterization of essential oil was evaluated using gas chromatography-flame ionization detector and gas chromatography-mass spectrometer analysis. Antibacterial activity of the oil was tested against four bacterial strains responsible for nosocomial infections: Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus and Citrobacter koseri using disc diffusion method. Results: Thirty three components were identified representing 89.30% of the total oil composition. The yield of essential oil was 0.4% and the predominant components were citronellal (14.40%), isogeraniol (6.40%), geraniol acetate (10.20%), nerol acetate (5.10%), caryophyllene (8.10%) and b-caryophyllene oxide (11.00%). Antibacterial activity of the oil showed the higher activity against all bacterial strains tested. Conclusions: The essential oil extracted from lemon balm can be used to clean the environment of reanimation polyvalent and anaesthesia service.