Background: Dielectric barrier discharge (DBD), a source of non-thermal plasma, is used in surface decontamination. Objective: To study the effect of DBD plasma treatment, we evaluated the effect of plasma exposure time on inactivation of Bacillus subtilis. Results: Applying the DBD plasma to the culture of B. subtilis caused complete sterilization of the surface without any thermal effects. In addition, the inactivated colony-forming units increased as the exposure time rises. Conclusion: Considering the low temperature and non-destructive features of this method, it seems that this method is applicable for fast sterilization of resistant bacteria and hospital sensitive instruments.