The relationship between the consumption of fresh produce and reduced risk of cardiovascular diseases makes these products a necessary component of healthy diet. However, concerns have been raised because of increased disease outbreaks associated with the consumption of contaminated produce. The development of new and promising decontamination technologies is necessary to provide safe produce to the public and to prevent economic losses. The objectives of this study were (i) to assess the efficacy of gaseous ozone in combination with vacuum cooling for the inactivation of Escherichia coli K12 and E. coli O157:H7 spot inoculated on leaves of baby spinach and Romaine lettuce, and (ii) to evaluate the efficacy of a pre-washing step, integrated into the gaseous ozone treatment, to enhance the inactivation of E. coli K12 and E. coli O157:H7 on baby spinach and cut Romaine lettuce leaves.The efficacy of gaseous ozone in combination with vacuum cooling was evaluated for inactivation of Escherichia coli on ready-to-eat baby spinach and Romaine lettuce leaves. In a preliminary experiment, baby spinach leaves were spot inoculated with E. coli K12. Inoculated leaves were vacuum cooled (-28.5 in. Hg) and then treated with gaseous ozone at the following conditions: 1.5 g/kg ozone in gas mix at 10 psig holding pressure for 30 min holding time. Compared to inoculated non-treated samples, oxygen only (control) and gaseous ozone treatment decreased the microbial populations 0.27 and 1.29 log CFU/g, respectively. Similarly, baby spinach leaves were spot inoculated with E. coli O157:H7 and treated at the same conditions described in the preliminary study. Efficacy of the treatment varied with the crop season. Reductions on baby spinach were 1.86 and 1.59 log CFU/g of E. coli 0157:H7 in January, 2011 and February, 2012 respectively, and these reductions were significantly higher than the average 0.97 log reduction obtained between May and September, 2011. The color of baby spinach leaves was comparable to non-treated produce. The same gaseous ozone treatment of hearts of Romaine yielded a reduction of 1.47 log CFU/g of E. coli O157:H7, however discoloration of the produce was evident. The effect of a fan in the treatment vessel also was evaluated. Fan-off treatments decreased inactivation of E. coli O157:H7 significantly compared to fan-on treatments.The effect of pre-washing and gaseous ozone treatment for the inactivation of E. coli on baby spinach and Romaine lettuce was also evaluated. Baby spinach and Romaine lettuce leaves were dip-inoculated with E. coli K12 and washed with 1% dimethyl sulfoxide (DMSO) or sterile distilled water. Washed leaves were treated with gaseous ozone, with initial vacuum application (-28.5 in. Hg), at the following conditions: 2.0 g/kg ozone at 10 psig holding pressure for 30 min holding time. There was no significant difference between washing with water and washing with DMSO in terms of microbial inactivation. The various washing treatments did not show any significant difference from each other, nor were the combination treatments different from one another. However, the combination gave significantly more reduction in the target from any washing alone treatments. Based on the results obtained from this set of experiments, a new washing equipment was constructed. At the same washing and gaseous ozone treatment conditions, efficacy of sterile distilled water, 2% PRO-SAN, and 200 ppm chlorine, alone or in combination with gaseous ozone, were evaluated. Chlorine or PRO-SAN alone decreased the microbial population 1.1 and 1.2 log, respectively. Chlorine and PRO-SAN followed by gaseous ozone treatment decreased the E. coli O157:H7 population 2.3 and 2.1 log, respectively, and microbial reductions were significantly higher than any other treatment. Environmental and health concerns related to chlorine increase the appeal of using PRO-SAN alone or in combination with gaseous ozone for the production of microbiologically safe, fresh produce.