The effect of freezing temperature (−7, −15, −30 and −80°C), number of freeze/thaw cycles (1 to 5 cycles) and sample volume (100 mL and 100 µL) on the viability of a pathogenic and an opportunistically pathogenic Escherichia Coli, a vancomycin‐resistant and a vancomycin‐sensitive Enterococcus Faecalis were examined. About 3.3 to 4.3 and 1.5 to 2.4 log reduction in cell density were observed in E. coli and E. faecalis, respectively, in the 100 mL samples frozen at −30°C or warmer. Freezing at −80°C was the least effective in killing the microbes, on average the log reduction at −80°C was approximately 1.0 to 1.5 units less than those achieved at the three warmer temperatures. Based on statistical analysis, cell inactivation levels achieved at −7, −15, or −30°C were not significantly different (P‐value = 0.1648). There were no statistical difference in terms of log reduction obtained under all experimental conditions for the two E.coli strains (P‐value = 0.46) and the two E. faecalis strains (P‐value = 0.10). The number of freezing/thaw cycles and sample volume, however, profoundly affected inactivation capacity of freezing. Freezing could be an effective method for further reduction of E. coli and Enterococcus in municipal wastewater/sludge.