To investigate the phenolic composition, content, and biological activities of highland barley fermentation spent (HBFS), phenolic compounds were extracted from highland barley and HBFS obtained at different fermentation stages. Total phenolic content was determined, individual phenolic compounds were identified, and differences in antioxidant and antifungal activities among samples were evaluated. Results showed that the total phenolic content of HBFS ranged from 4.72 to 11.25 mg•g-1. A total of 22 phenolic compounds were identified, with ferulic acid, catechin, gallic acid, syringic acid, rutin, p-coumaric acid, and chlorogenic acid as the predominant constituents. Discarded HBFS exhibited the highest total phenolic content (11.25 mg•g-1) and the highest total detected phenolic concentration (1 470.64 μg•g-1). Both free and bound phenolic extracts from discarded HBFS demonstrated strong antioxidant and antifungal activities. The DPPH and ABTS+ radical scavenging capacities and FRAP reducing power of free phenolics reached 2 052.72, 2 319.20, and 5 050.71 μg TE•g-1, respectively, while those of bound phenolics were 1 996.89, 1 710.69, and 3 344.82 μg TE•g-1, respectively. Antifungal assays showed inhibition zone diameters of 15.55 and 13.07 mm against Aspergillus niger and 36.56 and 37.78 mm against Penicillium spp. for free and bound phenolics, respectively. The minimum inhibitory concentration and minimum fungicidal concentration for both extracts were 1.56 and 3.13 mg•mL-1, respectively. These findings demonstrate that discarded HBFS is a rich source of phenolic compounds with pronounced antioxidant and antifungal activities, indicating its strong potential for development as a natural preservative or functional ingredient in food and pharmaceutical applications.