Neospora caninum is an important cause of bovine abortion worldwide for which dogs are the definitive host. The present study was aimed at investigating the exposure to N. caninum infection based on lifestyle categories of dogs from southern Romania. For this purpose, randomly selected rural and urban dogs were examined for fecal N. caninum-like oocysts and were serologically tested for the presence of anti-N. caninum IgG antibodies. Of the 386 dog fecal samples, N. caninum-like oocysts were found in 19 (4.9%; 95% CI = 2.89–7.59) as follows: rural guard dogs (4/41; 9.8%), cattle farm dogs (6/118; 5.1%), and stray dogs (9/192; 4.7%) (P > 0.05). None of the 35 urban guard dogs was positive. Serum samples (n = 84) from all of the 19 N. caninum-like oocysts-positive dogs and another 65 randomly selected canines (15 cattle farm dogs, 21 rural guard dogs, and 29 strays) were tested by indirect fluorescent antibody test (IFAT). None of these dogs exhibited any symptoms of clinical neosporosis. However, IgG antibodies against N. caninum were detected in 17/84 (20.2%) (P < 0.05) serum samples. The highest prevalence was registered in cattle farm dogs (38.1%) followed by strays (18.4%) and rural guard dogs (8.0%). The seropositivity to N. caninum increased significantly with age (P < 0.05), reaching 66.7% in dogs >10 yr of age, suggesting post-natal exposure to N. caninum is the predominant mechanism of N. caninum recruitment.