BACKGROUND Culture-negative Bartonella quintana endocarditis is challenging to diagnose and is associated with high mortality rates. Diagnostic confirmation of Bartonella quintana infection requires specialized assays, as identifying Bartonella henselae endocarditis by serology can be difficult due to the high rate of serological cross-reactivity. This is a case report of culture-negative Bartonella quintana endocarditis that was diagnosed with epidemiologic data, histology, and nucleic acid amplification testing. CASE REPORT A 28-year-old man with a history of homelessness was admitted to hospital with worsening productive cough, weight loss, and abdominal pain. A transthoracic echocardiogram (TTE) showed pulmonary valve vegetation and several aortic valve vegetations. His hospital course was complicated by cardiogenic shock and septic shock requiring transfer to a tertiary care medical intensive care unit. Although blood cultures remained negative for bacterial infection, serology testing was positive for Bartonella henselae and Bartonella quintana IgM and IgG. Nucleic acid amplification testing for 16S ribosomal RNA (rRNA) using valve tissue was diagnostic for Bartonella quintana. CONCLUSIONS This case of culture-negative Bartonella quintana endocarditis demonstrates the use of diagnostic nucleic acid amplification methods to confirm the diagnosis.