The Sustainable Sanitation System is a new wastewater treatment system that incorporates a non-flushing toilet (Bio-toilet) that converts excreta into a reusable resource (as fertilizer or humus for organic agriculture) and reduces the pollution load to environments of the rivers, the lakes, and the sea. However, the risk of exposure to pathogens should be considered, because excrement is stored in the Bio-toilet. The aim of the present work is to analyze the health risk of dealing with the matrix (excreta and urine mixed with sawdust) of the Bio-toilet. Therefore, the fate of pathogenic viruses was investigated using coliphages as a virus index, and the modeling of the die-off rate in matrix was introduced. Then the microbial risk assessment was applied to a Bio-toilet that was actually used in a residential house; the infection risks of rotavirus and enterovirus as reference pathogens were calculated. According to the lab-scale experiment using coliphages for investing the die-off rate of viruses in the Bio-toilet, Qβ had a higher die-off, which was greatly influenced by the water content and temperature. On the other hand, T4 showed a lower rate and was independent of water content. Therefore, these two phages' data were used as critical examples, such as viruses having high or low possibilities of remaining in the Bio-toilet during the risk assessment analysis. As the result of the risk assessment, the storage time required for an acceptable infectious risk level has wide variations in both rotavirus and enterovirus cases depending on the phage that was used. These were 0–260 days' and 0–160 days' difference, respectively.