The lactic acid bacteria are one of the sustainable ways of food production. As the native lactic acid bacteria (LAB) easily manipulate the substrate, helps in production of health essential probiotics with enhancing the bioavailability of the substrate. Here also, in present study, the native LAB isolates isolated from the millets and characterize them for the functional analysis for the human health association. In the present study, fermented millet-associated lactic acid bacteria were screened and characterized for their probiotic potential, safety evaluation and antimicrobial activity. A total of 33 isolates were purified as lactic acid bacteria based on colony shape and biochemical assays. However, only 13 isolates were found to be catalase-negative. Among the 13 isolates, 5 isolates exhibited optimum growth at 6.5% and 9.5% of salt concentrations, pH of 4.5 to 8.5 and 17 °C to 40 °C of the temperature. The probiotic properties of the five isolates exhibited that the survival rates in acid and bile salt concentration ranged from 56.2 to 73.7% and 55.3 to 70.3%, respectively. Similarly, the surface hydrophobicity of the isolates was 41-75%. Antibiotic assay revealed that all five isolates were resistant to Amoxicillin, Cloxacillin, and Penicillin-V. Interestingly, all the isolates except ME26 displayed susceptibility towards Penicillin (2 units) and Tetracycline (10 µg). Further, the four isolates (ME25, ME26, ME9, and ME2) had more antifungal activity against Aspergillus flavus. However, only three, except ME1 and ME2, showed maximum antibacterial activity and produced more antimicrobial compounds compared to reference strain L. plantarum Pb3. The potential probiotic isolates were identified as Weisella cibaria ME9, Weisella cibaria ME26, and Weisella confusa ME25.