The research aimed to explore the potential probiotic characteristics ofLevilactobacillus brevisRAMULAB49, a strain of lactic acid bacteria (LAB) isolated from fermented pineapple, specifically focusing on its antidiabetic effects. The importance of probiotics in maintaining a balanced gut microbiota and supporting human physiology and metabolism motivated this research. All collected isolates underwent microscopic and biochemical screenings, and those exhibiting Gram-positive characteristics, negative catalase activity, phenol tolerance, gastrointestinal conditions, and adhesion capabilities were selected. Antibiotic susceptibility was assessed, along with safety evaluations encompassing hemolytic and DNase enzyme activity tests. The isolate's antioxidant activity and its ability to inhibit carbohydrate hydrolyzing enzymes were examined. Additionally, organic acid profiling (LC-MS) andin silicostudies were conducted on the tested extracts.Levilactobacillus brevisRAMULAB49 demonstrated desired characteristics such as Gram-positive, negative catalase activity, phenol tolerance, gastrointestinal conditions, hydrophobicity (65.71%), and autoaggregation (77.76%). Coaggregation activity againstMicrococcus luteus, Pseudomonas aeruginosa, andSalmonella entericaserovar Typhimurium was observed. Molecular characterization revealed significant antioxidant activity inLevilactobacillus brevisRAMULAB49, with ABTS and DPPH inhibition rates of 74.85% and 60.51%, respectively, at a bacterial cell concentration of 109 CFU/mL. The cell-free supernatant exhibited substantial inhibition of α-amylase (56.19%) and α-glucosidase (55.69%)in vitro.In silicostudies supported these findings, highlighting the inhibitory effects of specific organic acids such as citric acid, hydroxycitric acid, and malic acid, which displayed higher Pa values compared to other compounds. These outcomes underscore the promising antidiabetic potential ofLevilactobacillus brevisRAMULAB49, isolated from fermented pineapple. Its probiotic properties, including antimicrobial activity, autoaggregation, and gastrointestinal conditions, contribute to its potential therapeutic application. The inhibitory effects on α-amylase and α-glucosidase activities further support its anti-diabetic properties.In silicoanalysis identified specific organic acids that may contribute to the observed antidiabetic effects.Levilactobacillus brevisRAMULAB49, as a probiotic isolate derived from fermented pineapple, holds promise as an agent for managing diabetes. Further investigations should focus on evaluating its efficacy and safetyin vivoto consider its potential therapeutic application in diabetes management.