Neosporosis in cattle is a globally important disease, causing abortions and significant economic losses if epidemic abortions occur. In Serbia, however, studies of Neospora caninum infection in cattle are few and are based on limited samples and/or from limited areas. We thus performed a nationwide study to examine the seroprevalence of N. caninum infection in a sample of cows randomly selected from dairy farms in 12 epidemiological units from throughout Serbia, as well as the possible transmission risk factors. Sera from a total of 1496 cattle were tested by competitive ELISA, and N. caninum-specific antibodies were shown in 7.2% animals (95% confidence interval CI, 6.6-7.9%), ranging from 2.2 to 12% across the epidemiological units. At least one seropositive animal was detected on 10.7% (95% CI, 9.7-11.8%) of farms, with a range of 5.9-25.9%. Logistic regression analysis showed that the single risk factor for infection in individual animals was keeping cows in loose-stalls (OR = 3.31, 95% CI = 1.95-5.60, P < 0.001). Risk factors for the presence of infection on farms also included housing in loose-stalls (OR = 18.49, 95% CI = 5.40-63.36, P < 0.001), and herd size > 100 animals (OR = 24.08, 95% CI = 3.85-150.50, P = 0.001). In view of the relatively low prevalence of infection showed at both the individual and farm level, this is the perfect time to undertake appropriate preventive measures to improve animal health and reduce economic losses.