Enteric pathogens can be present in drinking water catchments due to several point and non-point sources of faecal contamination. Pathogen and contaminant signatures will decay due to environmental stresses, such as temperature, Ultra Violet (UV) radiation, salinity, and predation. In this study, we determined the decay of the culturable faecal indicator bacterium (FIB) Escherichia coli (E. coli), two sewage-associated marker genes (Bacteroides HF183 and crAssphage CPQ_056), and enteric pathogens (Campylobacter spp., human adenovirus 40/41, and Cryptosporidium parvum) in two freshwater laboratory microcosms using culture-based, quantitative PCR (qPCR) and vital dye (determine the fraction of viable Cryptosporidium oocysts) assays. Freshwater samples from the Lake Wappa and Lake Wivenhoe (Australia) were seeded with untreated sewage and C. parvum oocysts, and their declining concentrations were measured over a 28-day period. Moreover, 16S rRNA amplicon sequencing was also undertaken to determine the change/shift in sewage-associated bacterial communities using SourceTracker. Overall, culturable E. coli and the HF183 marker gene decayed significantly (p < 0.05) faster than did the qPCR measured enteric pathogens suggesting that the absence of culturable FIB or qPCR HF183 in water samples may not indicate the absence of pathogens. The decay of crAssphage was similar to that of HAdV 40/41 and other pathogens tested, suggesting crAssphage may be a better surrogate for enteric viruses in sub-tropical catchment waters. The decay rates were greater at 25 °C compared to 15 °C, suggesting that FIB and pathogens persist longer in the winter season compared to summer. Overall decay rates of the tested microorganisms in this microcosm study suggest that sub-tropical conditions, especially temperature, have a negative impact on the persistence of tested microorganisms. Sewage-associated bacterial communities also showed similar patterns. Based on the results, which showed differences in simulated summer and winter temperatures for pathogen decay, corresponding management options and treatment need to be adjusted accordingly to minimize human health risks effectively. Display omitted •The HF183 marker gene decayed significantly (p < 0.05) faster than pathogens.•The decay of crAssphage was similar to that of HAdV 40/41.•The decay rates of tested organisms were greater at 25 °C compared to 15 °C.•The persistence of microorganisms is affected by temperature.