Highland barley bran (HBB), a by-product of milling, is rich in bioactive compounds with significant health benefits. This study explores the bioavailability and bioactivity of bound phenolic compounds (BPCs) in HBB through different pretreatments, such as microwave (M; HBB-M), enzyme (E; HBB-E), and germination (G; HBB-G), before digestion, during digestion, and colonic fermentation. The results showed that the antioxidant activities and the release of BPCs across the different phases are observed in a decreasing order: prior to digestion > following digestion > after colonic fermentation. Moreover, the bioavailability of BPC in HBB was affected by the pretreatments, after gastrointestinal digestion, HBB-E liberated 10.90 ± 1.88% of BPC, and HBB-M, HBB-C, and HBB-G lost 7.14 ± 0.54%, 5.94 ± 0.83%, and 3.96 ± 0.22% of BPC, respectively, and after colonic fermentation, they liberated 64.23 ± 1.25%, 68.30 ± 0.14%, 53.50 ± 2.34%, and 59.41 ± 1.51% of BPC, respectively. The highest contents of extractable BPC, SDF, and SDF/IDF ratio were observed in HBB-M after 48 h of fermentation. Furthermore, the abundance of Bifidobacteriaceae, Veillonellaceae, Oscillospiraceae, and Lachnospiraceae in HBB-M, was significantly promoted, compared to that of other groups, and SCFAs, especially butyric acid and propionic acid, were significantly increased. The results of this study indicated that HBB after microwave pretreatment exhibits relatively higher BPC bioaccessibility, extractability, antioxidant activities, and SCFA production, thus offering a sustainable bioprocessing strategy for HBB valorization.