Objective To investigate the occurrence of Bartonella sp. infection in asymptomatic horses and donkeys living in Tuscany, Central Italy. Methods Blood samples were collected from 77 horses and 15 donkeys and tested by indirect immunofluorescent test to detect antibodies against Bartonella sp. and by PCR to detect the pathogen. Results Fifty-four (58.69%; 95% CI: 47.95%–68.87%) animals, 9 donkeys and 45 horses, were seropositive with antibody titers ranging from 1:64 to 1:512. PCR assays detected 9 horses positive for Bartonella sp. and 3 donkeys for Bartonella henselae genotype I. Conclusions The detected sero-prevalence suggests a common and frequent exposure of equids living in Central Italy to bartonellae and PCR results show that Bartonella sp. infection is possible both in horses and donkeys. At the best of our knowledge, this is the first report of Bartonella henselae infection in donkeys.