The supercritical carbon dioxide (SC-CO2) process involves pressurizing CO2 in a chamber which generates liquid phase of carbon dioxide. Pressurized liquid CO2 has a strong extraction capability of organic and inorganic compounds. The recent studies have also demonstrated that antimicrobial effect of SC-CO2 due extraction some cellular components of microorganisms. The efficacy of a supercritical carbon dioxide treatment on alfalfa seeds contaminated with Escherichia coli K12 was tested at 2000, 3000, and 4000 psi at 50C. Samples were treated for 15, 30, and 60 min at each pressure. After pummeling the seed samples in 0.1% peptone water, the initial and final Escherichia coli and total aerobic bacteria on the seeds were determined by plating on 3M Petri Films. After 48 h of incubation at 37 C, the colonies were enumerated. Treated seeds were evaluated in terms of germination characteristics. For aerobic plate count, the effect of pressure in the range of 2000–4000 psi was not statistically significant (p > 0.05) even though 85.6% inactivation was achieved at 4000 psi for 60 min. For E. coli, the reductions for 2000, 3000, and 4000 psi treatments for 15 min were 26.6, 68.1, and 81.3%, respectively. As the time was increased from 15 to 60 min at 4000 psi, the percent E. coli reduction increased from 81.3% to 92.8%. The percent germination for all treatments was over 90%. There was no significant difference (p > 0.05) in the germination rate of treated and untreated seeds. Supercritical carbon dioxide treatments demonstrated a reduction of E. coli K12 and total aerobic counts without affecting the germination characteristics of alfalfa seeds (p < 0.05). This study was a step in the direction of improving safety of alfalfa seeds used to produce fresh sprouts, which have been the cause of several outbreaks.