Vibrio vulnificus and Vibrio parahaemolyticus are halophilic bacteria and can cause illness associated with the consumption of oysters. In this study, we tested the hypothesis to see if the adjustment of the acceleration voltage can change the efficacy of X-ray doses on V. vulnificus and V. parahaemolyticus in pure culture and inoculated V. parahaemolyticus within farm-raised live oysters (Crassostrea virginica). By using a tailor-made X-ray irradiator (Kimtron 350), the acceleration voltage was adjusted to 50, 200 or 350 kV and irradiated on pure culture of V. vulnificus or V. parahaemolyticus and farm-raised live oysters accumulated with a three-strain mixture of V. parahaemolyticus. Inactivation of V. vulnificus and V. parahaemolyticus pure cultures without filter had a lower inactivation when compared with filtered X-ray. With a 1 mm aluminum filter, 250–300 Gy X-ray reduced more than 6 log CFU ml−1 at 350 kV acceleration voltage. When 350 kV X-ray was applied on live oysters with the aluminum filter, 6 log MPN g−1 reduction of V. parahaemolyticus was achieved with 1250 Gy. The V. parahaemolyticus was significantly (p < 0.05) reduced in whole shell oysters from 8.11 ± 0.19 to 6.11 ± 0.19, 3.97 ± 0.47, 4.17 ± 0.17 and 2.77 ± 0.28 log MPN g−1 after processing with 250, 500, 750 and 1000 Gy, respectively. These results showed that the pathogen inactivation can be improved by the optimization of energy level with the adjustment of irradiation filter and acceleration voltage.