In Taiwan, the first human case of cat-scratch disease (CSD) was diagnosed by a serologic test in 1998. Since then, no studies have been conducted to understand the epidemiology of the infection in Taiwan. Therefore, this study is the first epidemiologic survey of CSD in cats and humans in this country. Using veterinary-associated individuals as the study population, it was identified that 1.7% of them were seropositive for B. henselae, and residence was the only factor associated with seropositivity. Bartonella species were successfully isolated from 25 (19.1%) of the 131 cats tested. Only B. henselae and B. clarridgeiae were obtained from bacteremic cats. Furthermore, 9.2% of 131 cats were dually-infected with genotypes I and II of B. henselae. It is the highest prevalence of co-infection that has ever been reported worldwide. In cats, the seroprevalence was 23.7% by indirect immunofluorescence antibody assay with B. henselae Houston-1 (type I) as the antigen. When 12 bacteremic but seronegative cats were re-tested by IFA slides coated with B. henselae U-4 antigen (type II), 9 cats were identified to be seropositive. Our study further suggested that using only direct PCR of 16S-23S rRNA intergenic region or the combination of the PCR method and indirect immuno-fluorescence test will be useful to diagnose Bartonella-free cats.