Blood culture-negative endocarditis (BCNE) is a challenging disease because of the significant impact of delayed diagnosis on patients. In this study, excised heart valves and blood serum samples were collected from 50 BCNE patients in two central hospitals in Tehran, Iran. Sera were tested by IFA for the presence of IgG and IgM antibodies against
Bartonella quintana
and
B. henselae
. Genomic DNA extracted from the heart valves was examined for
Bartonella
-specific
ssrA
gene in a probe-based method real-time PCR assay. Any positive sample was Sanger sequenced. IgG titer higher than 1024 was observed in only one patient and all 50 patients tested negative for
Bartonella
IgM. By real-time PCR, the
ssrA
gene was detected in the valve of one patient which was further confirmed to be
B. quintana
.
Bartonella
-like structures were observed in transmission electron microscopy images of that patient. We present for the first time the involvement of
Bartonella
in BCNE in Iran. Future research on at-risk populations, as well as domestic and wild mammals as potential reservoirs, is recommended.