Many factors have been mentioned as contributing to under-diagnosis and under-reporting of zoonotic diseases particularly in the sub-Sahara African region. These include poor disease surveillance coverage, poor diagnostic capacity, the geographical distribution of those most affected and lack of clear strategies to address the plight of zoonotic diseases. The current study investigates the knowledge of medical practitioners of zoonotic diseases as a potential contributing factor to their under-diagnosis and hence under-reporting. The study was designed as a cross-sectional survey. Semi-structured open-ended questionnaire was administered to medical practitioners to establish the knowledge of anthrax, rabies, brucellosis, trypanosomiasis, echinococcosis and bovine tuberculosis in selected health facilities within urban and rural settings in Tanzania between April and May 2005. Frequency data were analyzed using likelihood ratio chi-square in Minitab version 14 to compare practitioners' knowledge of transmission, clinical features and diagnosis of the zoonoses in the two settings. For each analysis, likelihood ratio chi-square p-value of less than 0.05 was considered to be significant. Fisher's exact test was used where expected results were less than five. Medical practitioners in rural health facilities had poor knowledge of transmission of sleeping sickness and clinical features of anthrax and rabies in humans compared to their urban counterparts. In both areas the practitioners had poor knowledge of how echinococcosis is transmitted to humans, clinical features of echinococcosis in humans, and diagnosis of bovine tuberculosis in humans. Knowledge of medical practitioners of zoonotic diseases could be a contributing factor to their under-diagnosis and under-reporting in Tanzania. Refresher courses on zoonotic diseases should be conducted particularly to practitioners in rural areas. More emphasis should be put on zoonotic diseases in teaching curricula of medical practitioners' training institutions in Tanzania to improve the diagnosis, reporting and control of zoonotic diseases. Veterinary and medical collaboration should be strengthened to enable more effective control of zoonotic diseases in Tanzania