Bartonella spp. are Gram-negative haemotropic bacteria transmitted by arthropod vectors. They are considered as important aetiologic agents of Blood Culture Negative Endocarditis (BCNE). The diagnosis of Bartonella endocarditis is often challenging because of its non-specific clinical presentation and difficulty in isolating the microorganism in culture using standard microbiological techniques as it is an intracellular bacterium. A combination of serological and molecular methods will aid in a comprehensive diagnosis of this condition. This study reports a series of four Bartonella endocarditis cases diagnosed in Chennai, Tamil Nadu, India. All of them were known Rheumatic Heart Disease (RHD) patients. The common valves affected in the patients were mitral and aortic valves. Three out of four cases showed presence of valvular vegetation in echocardiogram. One patient had a history of close contact with cats. Specific real time Polymerase Chain Reaction (PCR) targeting 16S-23S rRNA spacer region of Bartonella spp. was positive in three cases. Serology using Indirect Immunofluorescence Assay (IFA) was positive in one case. Western blot test was performed on serum samples of all four cases and Bartonella henselae was identified as the aetiological agent in one case. Extra cardiac complications were observed in three cases. Antibiotic treatment was successful in three out of four cases. One patient died due to acute renal failure.