Babesia is a tick-borne intraerythrocytic parasite that is clinically and diagnostically similar to malaria parasite, conferring risk of misdiagnosis in areas where both parasites are endemic. Data on Babesia in humans in Africa are lacking, despite evidence that it is present in regional animal populations. Samples that were collected in November 2014 to July 2015 in Kilosa district, Tanzania, were evaluated for evidence of malaria and Babesia infection. Clinical data and laboratory samples (i.e., hemoglobin, rapid diagnostic testing [RDT] for malaria, peripheral blood smear, and dried blood spots) from a routine survey were available for analysis. Dried blood spots were tested using an investigational enzyme linked immunosorbent assay (ELISA) against Babesia microti. A total of 1,030 children aged 1 month to < 5 years were evaluated; 186 (18.1%) were malaria RDT positive, 180 (96.8%) of whom had peripheral smears reviewed; 70/180 (38.9%) were smear positive for parasites. The median (inter quartile range) and range of B. microti ELISA signal to cutoff (S/C) ratio was 0.10 (0.06-0.15) and 0.01-1.65, respectively; the S/C ratios were significantly higher in subjects ≥ 1 year as compared with those < 1 year old (P < 0.001). There was also a statistically significant association between a positive RDT for malaria and the Babesia S/C (median 0.09 versus 0.13 in RDT negative versus RDT positive, respectively; P < 0.001). The highest S/C ratios were disproportionately clustered in a few hamlets. The findings suggest that Babesia may be present in Kilosa district, Tanzania. However, serological cross-reactivity and false positivity, notably between Babesia and Plasmodium spp., cannot be definitively excluded and have implications for testing in other settings.