Since the first report of Mycoplasma-associated polyarthritis in farmed Nile crocodiles in 1995, the disease has spread across Zimbabwe and South Africa and has resulted in significant economic losses on infected farms. Due to poor response to antimicrobial treatment and frequent relapses, the use of an autogenous vaccine to manage disease outbreaks was evaluated. Two previous trials had been performed with a similar vaccine and the results suggested that the vaccine could be effective in alleviating disease, although the numbers of animals were limited in both. This trial aimed to evaluate an inactivated, alum-adjuvanted M. crocodyli whole-cell vaccine in a large group of yearling crocodiles under field conditions on a farm in Zimbabwe where repeated M. crocodyli outbreaks have been reported. The safety of the vaccine was assessed by administrating the vaccine intraperitoneally to a subset of crocodiles. No adverse clinical reactions were observed in any of these crocodiles. A group of two thousand two hundred crocodiles received two intramuscular vaccinations four weeks apart in the autumn of 2011, while another group of two thousand two hundred crocodiles served as unvaccinated controls. Serum was collected from a subset of the vaccinated and unvaccinated crocodiles at different time-points before and after vaccination to evaluate the humoral response to vaccination. Latex slide agglutination tests (LAT) were performed on all samples and positive samples were titrated with the latex slide agglutination test and metabolism inhibition assay. A low percentage of sera were positive with serological tests done prior to vaccination, suggesting either circulating Mycoplasma or maternal immunity. Statistically significant increase in sero-positivity was detected with LAT four weeks after primary vaccination, although the titre remained low. Six weeks after the booster vaccination the percentage seropositive vaccinated crocodiles had decreased and there were no statistically significant difference between the percentage seropositive vaccinated and unvaccinated crocodiles. A significant outbreak of Mycoplasma-like polyarthritis was encountered 6 months after vaccination, in October 2011. Both vaccinated and unvaccinated crocodiles were affected. Serum samples from different subsets of crocodiles were collected and evaluated similar to the vaccine trial. The results indicated that a similar rate of sero-positivity was present in all crocodiles, irrespective of vaccination- or disease status Sera collected during this trial was used to evaluate the performance of the latex slide agglutination assay compared to the metabolism inhibition assay (“Gold standard” assay), as the performance of the LAT had not been evaluated previously. The calculated diagnostic sensitivity was 72%, diagnostic specificity was 32%, the predictive value of the positive test was 36% while the predictive value of the negative test was 69%. This trial indicated that the autogenous, inactivated, alum-adjuvanted, whole-cell vaccine against M. crocodyli was not able to protect farmed Nile crocodiles on an infected farm against clinical Mycoplasma-associated polyarthritis. It was also found that the latex slide agglutination assay could be useful as a robust, pen-side assay to evaluate exposure to M. crocodyli, although other assays, such as PCR, bacterial culture or growth inhibition assays, has to be performed to confirm the presence of disease.