The use of harvested rainwater in domestic hot water systems can result in optimised environmental and economic benefits to urban water cycle management, however, the water quality and health risks of such a scenario have not been adequately investigated. Thermal inactivation analyses were carried out on eight species of non-spore-forming bacteria in a water medium at temperatures relevant to domestic hot water systems (55–65 °C), and susceptibilities to heat stress were compared using D-values. The D-value was defined as the time required to reduce a bacterial population by 90% or 1 log reduction. The results found that both tested strains of Enterococcus faecalis were the most heat resistant of the bacteria studied, followed by the pathogens Shigella sonnei biotype A and Escherichia coli O157:H7, and the non-pathogenic E. coli O3:H6. Pseudomonas aeruginosa was found to be less resistant to heat, while Salmonella typhimurium, Serratia marcescens, Klebsiella pneumoniae and Aeromonas hydrophila displayed minimal heat resistance capacities. At 65 °C, little thermal resistance was demonstrated by any species, with log reductions in concentration occurring within seconds. The results of this study suggested that the temperature range from 55 to 65 °C was critical for effective elimination of enteric/pathogenic bacterial components and supported the thesis that hot water systems should operate at a minimum of 60 °C.