The effect of gamma radiation (0, 1, 3, and 5 kGy) on the shelf life of farmed rainbow trout (Oncorhynchus mykiss) fillets that were treated with sodium acetate and vacuum packaged and subsequently stored under refrigeration was studied by measuring microbiological, chemical, and organoleptic changes. Radiation affected populations of bacteria, namely, H2S-producing bacteria and Enterobacteriaceae (P < 0.05). Initial total viable counts of the control samples were ca. 4.41 log CFU/g, whereas the respective counts in samples irradiated at 1, 3, and 5 kGy were 3.08, 1.46, and ≤1 log CFU/g at day 1 of refrigerated storage. The maximum count of Enterobacteriaceae reached 2.29 and 1.45 log CFU/g at the end of storage for 1 and 3 kGy, respectively, but at a 5-kGy dose no growth of Enterobacteriaceae was observed. Of the biochemical indicators, thiobarbituric acid values for irradiated trout were higher than for nonirradiated fish (P < 0.05). Sensory evaluation (taste) showed a reasonable and good correlation with bacterial populations with storage time. The results revealed that radiation at a high dose (5 kGy) might induce lipid and protein oxidation, although the growth of microorganisms was inhibited. Therefore, radiation at a low dose (3 kGy) could be used to control the microbial and safety biochemical indices of O. mykiss for up to 4 weeks at refrigerator temperature without adverse effects on quality and acceptability.