In the current study, ten lactic acid bacteria (LAB) isolates exhibiting anti-α-glucosidase activity were isolated from fermented food. It is directed at novel supplementary diets to prevent/improve diet-induced carbohydrate metabolism disorders and related chronic diseases. Moreover, to evaluate their safety, functionality, and probiotic potential via in vitro simulated test conditions. From 16s-rRNA sequencing, Pediococcus acidilactici (NKUST 803, 845, 858), Lactobacillus plantarum (NKUST 817, 828, 851), Levilactobacillus brevis (NKUST 816, 855) and Lactobacillus acidophilus (NKUST 803, 863) were identified. The results showed that the isolates possessed anti-pathogenic activity, auto-aggregation ability, hydrophobicity (47.44–96.4%), and gastric acid-resistant activity (79–99.1%), which proved their potential for probiotics in nutraceuticals to render hypoglycemic activity or antidiabetic effects to the host positively. Among tested isolates, L. plantarum 817 and P. acidilactici 858 exhibited maximum α-glucosidase inhibitory (AGI) activity of 35–40%. The heat map clearly showed that L. plantarum 817 exhibited the best AGI activity and probiotic potential, among others. These were studied under various simulated gut conditions and safety tests. However, all isolates possess the potential to be used as probiotics in commercial-scale health applications. Pediococcus sp. possesses notable AGI activity but relatively less colonization potential in the gut hence recommended daily intake for positive health effects.