In studies of nematode-suppressive soil and plant-parasitic nematode management, it is often desirable to kill nematodes in the soil but keep the microbial community alive. The effect of temperature treatment on survival of Heterodera glycines, the soybean cyst nematode (SCN), and associated fungi and bacteria was investigated. Extracted eggs and cysts, and cysts in soil were subjected to treatments of temperatures ranging from −80 to 55°C for 1 to 24 h. Nematode survival was determined by hatching in vitro and by infectivity and development in soybean, and fungal survival was determined by plating the eggs or cysts on potato dextrose agar. The nematodes survived well between −20 and 45°C, but could not survive at −80 or 48°C and above, while some nematodes were killed after heat treatment of 46°C. By contrast, fungi survived well in the cysts at −80°C, and also much better than nematodes at the high temperatures. Glasshouse studies demonstrated that both bacteria and fungi survived well in the soil treated with −80°C. Transferring 5% of −80°C-treated soil to autoclaved soil could effectively establish nematode suppressiveness. Our study demonstrated that freezing soil at −80°C can kill SCN but maintain nematode suppressiveness, and the soil treatment can improve the method for nematode-suppressive soil evaluation.