The inactivation rates of Escherichia coli (E. coli) and MS2 phage were determined in fresh undiluted, diluted and six months aged stored urine samples at three temperatures ranging from 15-35°C in a subtropical region of Australia. In addition, Quantitative Microbial Risk Assessment (QMRA) calculations were undertaken to estimate the storage times that would be required to reduce the risk of infection by Campylobacter jejuni (using E. coli inactivation data) and rotavirus (using MS2 phage data) to an annual probability of infection of 10−4 during irrigation and consumption of lettuce. Higher inactivation rates were observed at a higher temperature (35°C) compared to lower temperatures (15 and 25°C) for both E. coli and MS2 phage. Stored urine sample also showed higher rates of inactivation for both E. coli and MS2 compared to undiluted and diluted urine samples at all temperatures. QMRA calculations indicated that inactivation of both bacteria and viruses to meet the health target of an annual probability of infection of 10−4 would take approximately four months at 15°C, 10 days at 25°C and five days of storage times at 35°C. The results also indicated that an increase in temperature has a more substantial effect on reducing storage time than varying the urine dilution for both E. coli and MS2 phage. Combining the QMRA-based approach with pathogen reduction interventions as presented in this study provides a range of management options for regulators, and may reduce barriers to the application of source-separated urine associated with long storage times.