This study aimed to explore the potential of various probiotic bacteria to produce bioactive peptides through milk fermentation and their effects on glucose and fat regulation. Twenty distinct strains were evaluated for their proteolytic activity on milk proteins, leading to the generation of various peptides. The antioxidant activity, alpha-amylase, and lipase inhibition of peptide extracts from fermented milk were assessed. The results showed that L. gasseri EB01 (L5) exhibited the highest proteolytic activity, producing the most amount of free amino acids (340.26 f 1.34 mu g/mL) compared to unfermented milk (Control) (100.01 f 1.68 mu g/mL). L. reuteri DSM 17939 (L11) demonstrated the highest radical scavenging activity (86.11 f 2.54%), indicating strong antioxidant properties. The peptide extract derived from B. coagulans GBI-30 (B4) displayed optimal alpha-amylase inhibition (34.26 f 1.67%), making it a promising alternative to acarbose. L. reuteri DSM 20016 (L12) exhibited the highest lipase inhibition (32.92 f 1.47%), comparable to the efficacy of orlistat at 100 mu g/mL, indicating potential for peptide extract in treating obesity by preventing fat absorption. This comprehensive in vitro study revealed that specific strains of probiotic bacteria in fermented milk can produce bioactive peptides with significant proteolytic activities, antioxidant capacities, alpha-amylase, and lipase inhibitory activities, offering promising natural alternatives for managing diabetes and obesity.