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The incidence of serious infections caused by multidrug-resistant gram-positive and gram-negative bacteria has been increasing rapidly worldwide despite advances in antibacterial therapy in the last two decades. Among multidrug-resistant gram-positive and gram-negative bacteria, methicillin-resistant Staphylococcus aureus, carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii are of great importance, because they have emerged as primary nosocomial pathogens in hospital outbreaks. In this study, we investigated whether melatonin has antibacterial effects against these microorganisms in vitro. The minimum inhibitory concentration of melatonin was determined using a standard microdilution method at 24 and 48 hr. Melatonin inhibited microbial growth at both 24 and 48 hr; but results showed that melatonin had antibacterial effects against these microorganisms after 48 hr of incubation in lower doses [concentrations between 31.25 to 125 μg/mL (0.13–0.53 mM)]. Also, it was determined that melatonin has a more potent antimicrobial effect on gram-negative microorganism. Among possible mechanisms, it is concluded that melatonin showed antibacterial effects by reducing intracellular substrates.