© 2016 Wiley Periodicals, Inc. The effects of UV-C radiation on the microbiological and chemical quality of hybrid Colossoma macropomum × Piaractus mesopotamicus fillets were examined daily over a period of 6 days. The samples were submitted to three different packaging conditions: (VP) vacuum packaging; (UV-L) VP+UV-C radiation at lower dose (55.83 mJ/cm2); or (UV-H) VP+UV-C radiation at higher dose (160.97 mJ/cm2). The microbial groups examined presented lower growth rate as well as lower number of colonies in the stationary phase in samples submitted to UV-L and UV-H when compared to VP. The UV-C radiation (UV-L and UV-H) accelerated TVB-N and improved biogenic amine production, while preventing a major production of ammonia in fish samples. The results of the current study suggest that UV-C radiation enhanced the shelf-life of Colossoma macropomum × Piaractus mesopotamicus fillets by at least 50% by slowing down microbial growth parameters and delaying chemical changes. Practical Applications: Nonthermal technologies are being applied as a viable alternative to thermal techniques, frequently used in processing of fresh food products. UV-C radiation is a new nonthermal technique that can lead to superficial decontamination of fresh fish fillets and presents low-cost as well as easy implementation in food processing industries. This technology does not produce any reactivity, chemical waste or undesirable by-products. However, there are few data on the use of this technique in food processing, mainly in fish products. Thus, this study will provide key information for the control of the microbial growth and chemical compounds production originated during vacuum-packed fish spoilage.