Mycoplasma hominis endocarditis is extremely uncommon and difficult to diagnose. Atypical growth characteristics in routine bacterial culture and an inability to demonstrate the organism using Gram staining can lead to a delayed diagnosis of Mycoplasma hominis infections, and the organism is often missed. This report describes a patient with Mycoplasma hominis prosthetic valve endocarditis. The microorganism was recovered from the mitral prosthesis but was missed in blood cultures. This finding suggests that Mycoplasma hominis should be considered in the differential diagnosis of culture-negative endocarditis.