Pythium root rot is an economically important disease threatening greenhouse production of tobacco seedlings. Although methyl bromide was historically used for tray sanitation, the phase-out of the fumigant from agricultural use has left few options for growers to produce disease-free transplants. Steam sanitation at 80°C for 30 min has shown control of disease caused by Rhizoctonia solani and has been adopted for use to manage Pythium spp. This study evaluates other steam temperatures and time durations to effectively manage Pythium spp. in float-tray systems. Naturally infested trays steamed at 63, 71, and 77°C for 30 min significantly reduced Pythium spp. from trays compared with TriSan wash and CC-15 dip treatments. Float trays inoculated with Pythium spp. that were steamed at 70 and 80°C for 2 h 30 min, respectively, also significantly reduced Pythium spp. survival. Other fungi, likely saprophytic or beneficial organisms, were not significantly impacted by any steaming treatment.