Commensal bacteria from the skin and mucosal surfaces are routinely isolated from patient samples and considered contaminants. The majority of these isolates are catalase-positive Gram-positive rods from multiple genera routinely classified as diphtheroids. These organisms can be seen upon Gram staining of clinical specimens or can be isolated as the predominant or pure species in culture, raising a priori suspicion of a possible involvement in infection. With the development and adoption of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), suspicious isolates are now routinely identified to the species level. In this study, we performed a retrospective data review (2012 to 2015) and utilized site-specific laboratory criteria and chart reviews to identify species within the diphtheroid classification representative of true infection versus contamination. Our data set included 762 isolates from 13 genera constituting 41 bacterial species. Only 18% represented true infection, and 82% were deemed contaminants. Clinically significant isolates were identified in anaerobic wounds (18%), aerobic wounds (30%), blood (5.5%), urine (22%), cerebrospinal fluid (24%), ophthalmologic cultures (8%), and sterile sites (20%). Organisms deemed clinically significant included multiple Actinomyces species in wounds, Propionibacterium species in joints and cerebrospinal fluid associated with central nervous system hardware, Corynebacterium kroppenstedtii (100%) in breast, and Corynebacterium striatum in multiple sites. Novel findings include clinically significant urinary tract infections by Actinomyces neuii (21%) and Corynebacterium aurimucosum (21%). Taken together, these findings indicate that species-level identification of diphtheroids isolated with a priori suspicion of infection is essential to accurately determine whether an isolate belongs to a species associated with specific types of infection.