Frozen chicken breast was hydrolyzed by treatment with thermolysin enzyme to obtain a chicken hydrolysate containing bioactive peptides. After that, a peptide was purified from the chicken hydrolysate utilizing a Sep-Pak C18 cartridge and reversed-phase high-performance liquid chromatography (RP-HPLC). The molecular weight of the chicken peptide was 2766.8. Protein sequence analysis showed that the peptide was composed of 25 amino acid residues. The peptide, designated as C25, demonstrated an inhibitory action on the angiotensin-converting enzyme (ACE) with a half maximal inhibitory concentration (IC) value of 1.11 µg/mL. Interestingly, C25 showed antimicrobial activity against multi-drug resistant bacteria F24B and JM109, both with MIC values of 24 µg/mL. The chicken hydrolysate showed antioxidant activity with an IC value of 348.67 µg/mL. Furthermore, the proliferation of aerobic bacteria and as well as lipid oxidation were significantly reduced when the chicken hydrolysate was used as a natural preservative during cold storage of chicken breasts. Hydrolysates derived from muscle sources have the potential to be used in formulated food products and to contribute positively to human health.