Purpose Septic arthritis caused by Mycoplasma is rare. The diagnosis and effective treatment of mycoplasmal septic arthritis remains a serious problem for clinicians. The aim of this systematic review was to document the available evidence on the diagnosis and treatment methods for mycoplasmal septic arthritis and to provide guidance for clinicians. Methods The PubMed, EMBASE, and Cochrane Library databases were searched in December 2018.The searches were limited to the English language. Article screening and data extraction and compilation were conducted by two independent reviewers. All the included studies were assessed using the Methodological Index for Non-randomized Studies (MINORS) tool. Results There was a total of 33 articles including 34 cases of mycoplasmal septic arthritis and eight of them were periprosthetic joint infection (PJI). Twenty-four patients (70.6%) were immunocompromised, and the synovial fluid white blood cell (WBC) count was significantly lower in the immunocompromised group than in the immunocompetent group (48,527 × 10 6 /L vs. 100,640 × 10 6 /L; P = 0.009). The traditional culture method took longer, and the positivity rate was lower than that of nucleic acid testing (50% vs. 100%; P = 0.016). Only 19.2% (5/26) of patients treated with empiric antibiotics were relieved of symptoms, while 82.4% (28/34) of patients achieved satisfactory results after being treated with antibiotics against Mycoplasma. Conclusion The possibility of mycoplasmal septic arthritis should be considered if patients with joint infections have a history of immunocompromised, repeated negative cultures, and poor empiric antibiotic treatment results. The rational use of nucleic acid testing technologies can help in the clinical diagnosis and treatment of mycoplasmal septic arthritis.