Toxoplasma gondii and Neospora caninum are obligate intracellular intestinal coccidia distributed worldwide, and are causative agents of toxoplasmosis and neosporosis, respectively. The aim of this study was to evaluate the prevalence of anti-T. gondii and anti-N. caninum antibodies and the factors associated with infections in beef cattle intended for human consumption in an Amazonian area of North Brazil. We collected blood samples of 387 cattle from 50 herds located in different municipalities of the State of Rondônia. An epidemiological questionnaire was distributed to farmers, with regard to nutritional, sanitary and reproductive herd management. The samples were identified, refrigerated and sent for serological analyses via IFAT (Immunofluorescent Antibody Test). Among the 387 analyzed animals, 91 (23.5%; CI 95%: 18.8–27.2) were positive for anti-T. gondii antibodies, with titers varying from 1:64 (75.8%) to 1:512 (2.2%). For anti-N. caninum antibodies, only four animals (1%; CI 95%: 0–2.7) were positive, with titers ranging from 1:400 (50%) to 1:1600 (25%). We observed a significant rate of anti-T. gondii antibodies in the variables “pure breed” and “contact with free-range chickens” (p < 0.2). There were no risk factors associated with the presence of anti-T. gondii or anti-N. caninum antibodies. In conclusion, there was a high prevalence of anti-T. gondii antibodies in beef cattle intended for human consumption in the State of Rondônia, Brazil, and a low prevalence of anti-N. caninum antibodies. Longitudinal studies can better elucidate the cause of these prevalence levels and how they could be better prevented and controlled.