The aim of this study was to identify and characterize the bioactive peptide profile of Podolica cow's milk. This dairy product is known for its nutritional properties related to the presence of peculiar lipids and is a typical breed traditionally reared in southern Italy. Using top-down peptidomics, we identified 2213 peptides in milk samples from four different farms, with 19 matching bioactive sequences. Bioactivities include dipeptidyl peptidase-IV (DPP-IV) inhibition, angiotensin-converting enzyme (ACE) inhibition, antioxidant activity, enhanced calcium uptake, and other peptides with potential antimicrobial effects. DPP-IV-inhibitory peptides (e.g., LDQWLCEKL and VGINYWLAHK) suggest potential for type 2 diabetes management, while ACE inhibitors (such as YLGY and FFVAPFPEVFGK) could support cardiovascular health by reducing hypertension. Antimicrobial peptides such as SDIPNPIGSENSEK and VLNENLLR showed broad spectrum of activity against various harmful microorganisms, positioning Podolica milk as a promising source for natural antimicrobial agents. Additionally, peptides with osteoanabolic, antianxiety, and immunomodulatory properties further highlight the multifaceted health benefits associated with this type of milk. Our findings underline the functional richness of Podolica milk peptides with various bioactivity properties, which could enhance the value of derived dairy products and contribute to sustainable agricultural practices. Future research will aim to explore these bioactivity properties in vivo, establishing a foundation for functional foods and supplements based on Podolica milk.