The identification of antioxidant, antidiabetogenic and antimicrobial peptides obtained from sorghum spent grain (SSG) protein hydrolysate was performed. Antioxidant activity (AOA) was evaluated by ABTS+ radical cation scavenging, antidiabetogenic activity (ADA) by dipeptidyl peptidase IV (DPP-IV) inhibition, and antimicrobial activity (AMA) through the minimum inhibitory concentration (MIC) method against 2 Gram-negative and 3 Gram-positive bacteria. SSG peptides were fractionated using a Sephadex G-25 molecular exclusion column, and fractions collected (F1–F6) were evaluated through AOA, ADA and AMA assays. The chromatographic process allowed increasing 20% the AOA and 35% the ADA in F2, and 90% the AMA in fractions F5 and F6. The most active fractions (F2, F5 and F6) were evaluated using LC-ESI-Q-TOF tandem mass spectrometry, which allowed identifying six new peptides with high bioactivity. Moreover, the docking refinement protocol showed that identified peptides could interact with the DPP-IV enzyme, two of them in a competitive inhibition mode. Sorghum spent grain (SSG) could be an important source of bioactive peptides. Additionally, this is the first report indicating the presence of antibacterial peptides encrypted in sorghum proteins.