Neospora caninum plays a significant role in causing abortion and reproductive failure in dairy cattle. The majority of neosporosis-related abortions take place during the 5–6 months of gestation. Fetal death in the uterus, resorption, mummification, autolysis, stillbirth, birth with clinical symptoms, or being born clinically healthy but with chronic infection are all possible outcomes. The objective of the study was to identify N. caninum infection in aborted bovine fetuses through molecular analysis and mouse bioassay testing. From 2019 to 2022, 121 bovine aborted fetuses were collected from dairy farms in Khorasan Razavi province. The fetal brain samples were screened for detection of the parasite DNA by polymerase chain reaction assay (PCR). In addition, a portion of PCR-positive brain tissue was homogenized and inoculated into the peritoneum of five BALB/c mice. All mice were sacrificed six weeks post infection and examined using serology, microscopic, and PCR methods. If the mice's brain samples were PCR positive, the mouse bioassay test was repeated two times. The N. caninum DNA was detected in 19.8% of brain samples in bovine aborted fetuses. Among PCR-positive brain samples, only ten samples were suitable for mouse bioassay examination. All inoculated mice were seronegative without clinical signs after three times bioassays, although the brain samples of three mice groups were PCR-positive after repeated bioassays. The PCR results showed a moderate frequency of Neospora infection in aborted bovine fetuses. Furthermore, the isolates obtained in this study had low pathogenicity in BALB/c mice. It seems the isolates belong to an avirulent strain