Background Abiotrophia species have rarely been implicated in osteoarticular infections. We report one case of an A. defectiva knee prosthesis infection. Case presentation A 71-year-old man of Italian origin presented with pain and swelling of the knee four years after the implantation of a total knee replacement prosthesis. While standard culturing of the synovial fluid resulted in no isolation of microorganisms, the direct inoculation of the synovial fluid into a rich culture medium resulted in the identification of A. defectiva by polymerase chain reaction sequencing. Repeated attempts of culturing microorganisms from blood were negative, and echocardiograms and colonoscopies were unremarkable. High-dose amoxicillin for nine months and a two-stage replacement of the knee prosthesis led to full patient recovery by the time of the 12-month follow-up examination. Conclusions Because Abiotrophia spp. are fastidious microorganisms, it is likely that cases of Abiotrophia orthopedic infection are misdiagnosed as culture-negative infections. Direct inoculation of synovial fluids into rich broth medium and further polymerase chain reaction-based detection of culture-negative synovial fluids are key tests for accurate documentation and detection of these infections.