Mycoplasma bovis in cattle is difficult to diagnose. Recently, the ID screen® mycoplasma bovis indirect ELISA (ID screen) was commercially released by IDVet. The objectives of this study were to: (1) gain and share experience of using the ID screen in adult dairy cows under field conditions; (2) determine the correlation between antibody levels in milk and serum and (3) compare the ID screen results with those of the Bio K 302 (BioX 302) ELISA from BioX Diagnostics. Paired serum and milk samples were collected from 270 cows from 12 Danish dairy herds with three categories of M. bovis disease history. The ID screen tested nearly all cows positive in all, but the three non-infected herds, while the BioX 302 tested very few cows positive. The ID screen is therefore a much more sensitive test than the BioX 302. However, cows in five exposed herds without signs of ongoing infection and two herds with no history of M. bovis infection also tested ID screen positive. Therefore, the performance and interpretation of the test must be investigated under field conditions in best practice test evaluation setups. A concordance correlation coefficient of 0.66 (95% CI: 0.59–0.72) between the ID screen serum and milk results indicates that milk samples can replace serum samples for the ID screen diagnosis of M. bovis in adult cows.