Toxoplasmosis and neosporosis are parasitic diseases of livestock globally. In this study, we investigate the presence of Neospora caninum and Toxoplasma gondii in 250 beef cattle from Kanchanaburi, Ratchaburi and Nakhon Patom provinces, Thailand comparing with the results from 5 years earlier. The serological diagnosis for toxoplasmosis and neosporosis were performed by indirect ELISA technique using recombinant antigens TgGRA7 and NcSAG1, respectively. Furthermore, risk factors at the animal-and herd-levels were also evaluated. Seroprevalence of T. gondii, N. caninum and co-infections were 15.2%, 20.8% and 27.2%, respectively. In comparison to the results from 5 years earlier, seropositivity of N. caninum was 3.5 times higher (20.8% and 5.9%), while T. gondii antibody level was lower (15.2% and 25.7%). Large herd size and communal farming practice contributed to the risk of T. gondii and N. caninum infections in the cattle farm. This study provides information on the prevalence and risk factor of toxoplasmosis and neosporosis among beef cattle in the study areas and highlight the need for effective strategies to prevent bovine neosporosis in beef herds.