Orthopedic and trauma device-related infections (ODRI) due to high virulence microorganisms are a devastating complication after orthopedic surgery. Coagulase-negative Staphylococci (CoNS) are mainly involved but commensal bacteria, located in human mucous membranes, are emerging pathogens in ODRI. Currently, bacterial culture is the gold standard for ODRI but the diagnostic process remains time consuming and laborious. We evaluated a combination of microbiological approaches in the diagnosis of emerging pathogens involved in ODRI. We analyzed two synovial fluids, five tissue samples and five surgical wound swabs from two different patients with ODRI, attending the Department of Orthopedic and Trauma Surgery of Mater Domini Teaching Hospital, Catanzaro, Italy. Identification was carried out with a combination of microbiological approaches (culture, mass spectrometry and 16s rRNA gene sequencing). We demonstrated the importance of a combination of microbiological approaches for the diagnosis of emerging pathogens in ODRI, because the low number of cases in the literature makes it very difficult to formulate guidelines for the management of patients.