Tuberculosis is a zoonotic disease that mainly causes respiratory infection. However, it can also infect other organs such as the kidneys and bladder, which can lead to high counts of the organisms in the urine. Introducing urine diversion systems and reuse of the urine in agriculture may introduce new transmission routes for infection, increasing the risk of spread. This study evaluated the inactivation rate of mycobacteria in human urine for ensuring safe reuse in agriculture and examined whether current World Health Organization recommendations on storage time are sufficient for inactivating Mycobacterium tuberculosis and Mycobacterium bovis. In this study, a decimal reduction in M. tuberculosis and M. bovis in human urine containing 7 and 3 g NH(3)-N L(-1), respectively, was obtained in just over 10 days at 4°C and below three days at 22°C. This is considerably faster than previously reported reduction rates of mycobacteria in animal slurry at similar temperatures. Based on the present results, a storage time of five weeks at temperatures below 20°C or of two weeks at temperatures above 20°C is sufficient to prevent transmission of mycobacteria when recycling human urine. These values lie within the WHO recommended storage period.