We studied the survival of Escherichia coli and enterococci populations in fecal samples of 7 host species after storage at –20 and –80 °C for 30 days. Composite fecal samples were collected from cows, chickens, horses, pigs, dogs, birds, and humans, and bacteria were enumerated before and after storage. Twenty-eight colonies of each bacterial species were typed before and after storage and the strains were assigned to different biochemical phenotypes (BPTs). A significant reduction in the number of E. coli was observed in all samples stored at –20 °C but in only 3 of those samples stored at –80 °C. However, the numbers of enterococci were similar in most stored samples (except cow and birds). The number and the distribution of E. coli and enterococci BPTs in fresh samples did not vary significantly from those stored at either temperature. Furthermore, the population structure of E. coli and enterococci did not change significantly after storage at –80 °C, this was always the case for those samples stored at –20 °C. We conclude that for those studies investigating E. coli or enterococci population structure, short-term storage (≤30 days) of fecal samples in a glycerol broth at –80 °C is a preferable option.