In recent years, consumer trends have been changing toward fresh food products such as fruit juice, vinegar, etc. that are a good source of bioactive components, high nutritional characteristics, and beneficial microorganisms. Blackthorn (Prunus spinosa L.) vinegar (BV) is one of these nutritious foods. The study aims to examine the efficacy of ultraviolet-C (UV-C) light applied by a modified reactor and ultrasonication on bioactive compounds (total phenolic, total flavonoid, ascorbic acid content, and antioxidant activity) of traditionally produced BV. Furthermore, the volatile organic compound (VOC) profile, hydroxymethylfurfural (HMF) content, cytotoxicity properties, and color were assessed. UV-C light and ultrasonication processes enriched most bioactive components, but these methods did not significantly improve ascorbic acid (p > 0.05) compared to pasteurization. Twenty-seven volatile compounds were analyzed in order to determine the VOC profile. As a result, thermal and nonthermal methods were found to affect the profile significantly (p < 0.05). No significant differences were detected in total soluble solids (4.70−4.77), titratable acidity (3.81−3.87), and pH (3.39−3.41) values. The anticarcinogenic activities of UV-C-treated BVs were more significant than others. Nonthermal treatments were generally better than pasteurization in maintaining and enriching the quality of BV. In this study, UV-C light and ultrasonication technology can be used as an alternative to traditional thermal techniques to improve the quality of BV.