This study aimed to determine the frequency of Babesia spp. infection in cattle, livestock farmers, and patients with acute febrile illness (AFI) from the Magdalena Medio region in Colombia using molecular and serological methods. PCR detected Babesia in 83.9 % (161/192) of cattle and 14.8 % (21/143) of farmers tested. Molecular analysis based on eight DNA sequences from the 18S rRNA identified Babesia bovis and Babesia bigemina in cattle and Babesia bigemina in farmers. There was no molecular detection in the patients with acute febrile illness; nonetheless, the serological test in the AFI population yielded 10.7 % (23/215) seropositivity for Babesia microti. Our findings suggest natural infection by this hemoparasite in this livestock region, and it is, therefore, essential to continue determining the role of this parasite as an etiological agent of diseases in the area, not only because of its veterinary relevance but also because of its zoonotic potential.