The focus of this study was to determine the efficacy of various chemicals in eliminating 2.04 to 3.23 log10 CFU/g of Escherichia coli O157:H7 from alfalfa seeds and to determine the survivability of the pathogen on seeds stored for prolonged periods at three temperatures. Significant (P ≤ 0.05) reductions in populations of E. coli O157:H7 on inoculated seeds were observed after treatments with 500 and 1,000 ppm of active chlorine (as Ca[OCl]2) for 3 but not 10 min and with ≥2,000 ppm of Ca(OCl)2 regardless of pretreatment with a surfactant. Treatment with 20,000 ppm of active chlorine failed to kill 2.68 log10 CFU/g of seeds. Acidified NaClO2 (500 ppm) was effective in reducing populations of the pathogen by >2 logs per g. Acidified ClO2 significantly reduced populations of E. coli O157:H7 on seeds at concentrations ≥100 ppm, and 500 ppm of ClO2 reduced the pathogen from 2.7 log10 CFU/g to 2H3OH, but there was a dramatic decrease in germination percentage. Treatment with 0.2% H2O2 significantly reduced populations, and the organism was not detected by direct plating after treatment with ≥1% H2O2. Significant reduction in population of E. coli O157:H7 occurred after treatment with 1% trisodium phosphate, 40 ppm of Tsunami and Vortexx, and 1% Vegi-Clean. A significant decrease in the number of E. coli O157:H7 on dry seeds was observed within 1 week of storage at 25 and 37°C, but not at 5°C. Between 1 and 38 weeks, populations on seeds stored at 5°C remained relatively constant. The pathogen was recovered from alfalfa seeds initially containing 3.04 log10 CFU/g after storage at 25 or 37°C for 38 weeks but not 54 weeks.