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Fever of unknown origin (FUO) can be determined by different conditions among which infectious diseases represent the main cause.A young woman, with a history of aortic stenosis, was admitted to our unit for a month of intermittent fever associated with a new diastolic heart murmur and splenomegaly. Laboratory tests were negative for infectious screening. The total body computed tomography (CT) scan excluded abscesses, occulted neoplasia, or lymphadenopathy.The transthoracic and transesophageal echocardiogram showed an aortic valve vegetation. Three sets of blood cultures were negative for all microorganisms tested. According to these findings, Bartonella endocarditis was suspected and the serology tests performed were positive. Finally, real-time polymerase chain reaction (RT-PCR) detected Bartonella henselae DNA on tissue valve.The patient underwent heart valve surgery and a treatment of Ampicillin, Gentamicin, and oral Doxycycline was prescribed for 16 days and, successively, with Doxycycline and Ceftriaxone for 6 weeks.After surgery and antibiotic therapy, patient continued to do well.Bartonella species are frequently the cause of negative blood culture endocarditis. Molecular biology techniques are the only useful tool for diagnosis. Valvular replacement is often necessary and antibiotic regimen with Gentamicin and either Ceftriaxone or Doxycycline is suggested as treatment.Echocardiogram and blood cultures must be performed in all cases of FUO. When blood cultures are negative and echocardiographic tools are indicative, early use of Bartonella serology is recommended.