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Abstract Background and Objective: Enterococci are Gram-positive members of human gastrointestinal flora,in Dairy products and environment. they have emerged as important causes of opportunistic nosocomial infections in recent years. In this study we aimed to investigat and compare the efficiency of MALDI-TOFmass spectroscopy method through Biochemical and Molecular methods for detecting Enterococcus faecalis and Enterococcus faecium. Materials and Methods:seventhy five clinical samples were collected for biochemical, molecular and mass spectroscopy investigations. Samples were treated with Esculin hydrolysis, Catalase, Pyrrolidonylaminopeptidase, 6.5% NaCl solution, motility, 0.04% Tellurite, L-Arabinose and Sorbitol. Using specific primesallele specific PCR was used.The samples were then analyzed by MALDI-TOF mass spectroscopy and Biotyper 3 software. Results:Enterococcus faecium andEnterococcus faecaliswere detected in thirty and forty two samples, respectively whereas three samples showed both bacterial infections. Using biochemical analysis, two E.faecium isolates were Arabinose negative and one E. faecalis isolates was Telliurite negative. All sampleswere showed correct bands in PCR results but twoof them didn't show clear bands(on agarose gel). In mass spectroscopy analysis all strains were correctly detected and well defined. Conclusion: According to our results, MALDI-TOF mass spectrometry in comparison with Molecular and Biochemical Methodscould be a reliable and accurate method that can easily and quickly identify and differentiate Enterococcus faecium and Enterococcus faecalisin clinical samples. Key words:Enterococcus faecalis, Enterococcus faecium, MALDI-TOFmass spectrometry,PCR