To apply UV-C as a non-heating sterilization method to increase the microbiological safety of fresh seedless watermelon products, reductions in E. coli and quality changes by treatment dose (0, 2, 4, 8, 14, 20 kJ/m2) were investigated. The pH, sugar content, and hardness of watermelon inoculated with E. coli were not significantly different according to the UV-C treatment dose, but the polyphenol content was significantly decreased compared to the controls (425.4 GAE μg/g F.W.). When treated with 2 and 4 kJ/m2, the lycopene content was 31.6 and 30.9 μg/g F.W., respectively, which was increased compared to the controls (28.5 μg/g F.W.). The arginine and citrulline content was also significantly increased compared to the controls. The number of E. coli was significantly decreased compared to the controls following UV-C treatment. Considering the degree of E. coli reduction, lycopene content, arginine content, citrulline content, and UV-C irradiation time, subsequent experiments were conducted by selecting a UV-C treatment dose of 2 kJ/m2. The results of confirming the degree of reduction in the number of E. coli colonies by a single treatment and combined treatment with UV-C 2 kJ/m2 and 70% ethanol showed that the combined treatment was most effective as colonies were decreased by 2.3 log CFU/g compared to the controls. Therefore, it is judged that UV-C 2 kJ/m2 radiation and combined treatment with 70% ethanol could be applied as a non-heating sterilization method for fresh watermelon slices.