Mycobacterial pathogens can be excreted in human urine by some infected individuals. High numbers of pathogenic mycobacteria in the urine could represent a new transmission route for mycobacterial infections if the urine is used for crop fertilization. In this study, the survival of spiked Mycobacterium aurum and M. fortuitum as fast-growing mycobacteria and M. avium and M. bovis as slow-growing mycobacteria were tested in urine. The tests were conducted in fresh (<1 day old) and stored human urine (>6 months old) at temperatures of 15 and 30 °C. The results indicate that these mycobacterial strains survived less than 2 weeks in stored urine at 30 °C with a pH value of around 9.0. Mycobacteria had the longest survival time, up to 6 weeks, in fresh urine stored at 15 °C. There were negative correlations between the increase in pH and the number of spiked mycobacteria in urine. In conclusion, if human urine is to be used for fertilization, it is advisable to store it for more than 6 weeks at least at 15 °C in order to prevent survival and subsequent exposure to pathogenic mycobacteria.