Probiotics are live microorganisms with various health benefits when consumed in appropriate amounts. Fermented foods are a rich source of these beneficial organisms. This study aimed to investigate the probiotic potential of lactic acid bacteria (LAB) isolated from fermented papaya (Carica papayaL.) throughin vitromethods. The LAB strains were thoroughly characterized, considering their morphological, physiological, fermentative, biochemical, and molecular properties. The LAB strain's adherence and resistance to gastrointestinal conditions, as well as its antibacterial and antioxidant capabilities, were examined. Moreover, the strains were tested for susceptibility against specific antibiotics, and safety evaluations encompassed the hemolytic assay and DNase activity. The supernatant of the LAB isolate underwent organic acid profiling (LCMS). The primary objective of this study was to assess the inhibitory activity of α-amylase and α-glucosidase enzymes, bothin vitroandin silico. Gram-positive strains that were catalase-negative and carbohydrate fermenting were selected for further analysis. The LAB isolate exhibited resistance to acid bile (0.3% and 1%), phenol (0.1% and 0.4%), and simulated gastrointestinal juice (pH 3–8). It demonstrated potent antibacterial and antioxidant abilities and resistance to kanamycin, vancomycin, and methicillin. The LAB strain showed autoaggregation (83%) and adhesion to chicken crop epithelial cells, buccal epithelial cells, and HT-29 cells. Safety assessments indicated no evidence of hemolysis or DNA degradation, confirming the safety of the LAB isolates. The isolate's identity was confirmed using the 16S rRNA sequence. The LAB strainLevilactobacillus brevisRAMULAB52, derived from fermented papaya, exhibited promising probiotic properties. Moreover, the isolate demonstrated significant inhibition of α-amylase (86.97%) and α-glucosidase (75.87%) enzymes.In silicostudies uncovered that hydroxycitric acid, one of the organic acids derived from the isolate, interacted with crucial amino acid residues of the target enzymes. Specifically, hydroxycitric acid formed hydrogen bonds with key amino acid residues, such as GLU233 and ASP197 in α-amylase, and ASN241, ARG312, GLU304, SER308, HIS279, PRO309, and PHE311 in α-glucosidase. In conclusion,Levilactobacillus brevisRAMULAB52, isolated from fermented papaya, possesses promising probiotic properties and exhibits potential as an effective remedy for diabetes. Its resistance to gastrointestinal conditions, antibacterial and antioxidant abilities, adhesion to different cell types, and significant inhibition of target enzymes make it a valuable candidate for further research and potential application in the field of probiotics and diabetes management.