The present study aimed to evaluate the stability of Human Adenovirus type 2 (HAdV2) and Murine Norovirus 1 (MNV-1) in surface freshwater samples stored at different temperatures. For HAdV2 the stability decreased with increasing temperatures (−80 > −20 > 4 > 22 °C). The time required to reach one log reduction in viral titers (T90) was similar among all the times and temperatures by different cell-culture based methods and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The HAdV2 stability decreased with the time of storage temperature and methods employed, aside from samples stored at 22 and 4 °C which showed the lowest T90 values (50 days). For MNV-1, the samples stored at 22 and −20 °C showed higher log10 decay values, followed by 4 and −80 °C; while genome persistence was ranked as −80 > −20 > 4 > 22 °C. The T90 values were lower for samples stored at 22 °C (33 days), followed by 4, −20 and −80 °C with 111, 100 and 333 days, respectively. The results indicate that, under laboratory storage conditions, freshwater samples should be kept at 4 °C and at −80 °C for short- and long-term periods, respectively. This study provided useful information about thermal and temporal stability of the enteric viruses regarding sample storage conditions.