Incidence of bovine babesiosis in Portugal is currently unknown. In this study, a first survey of Babesia bovis and Babesia bigemina infection in cattle was carried out using blood samples from 406 clinically healthy individuals from different districts from Central and Southern regions of Portugal and analyzed by indirect enzyme linked immunosorbent assay (iELISA) and nested polymerase chain reaction (nPCR). Overall, serological testing revealed that 79% and 52% of cattle were positive for B. bovis and B. bigemina antibodies, respectively, whereas nPCR testing detected 71% and 34% cattle infected with B. bovis and B. bigemina protozoan, respectively. This is the first report of the prevalence of B. bovis and B. bigemina in cattle obtained by serological and DNA analysis studies in Central and Southern regions of Portugal. These data suggests high incidence of Babesia sp. infection in Portugal and can be used for designing adequate control programs.