The search for probiotic candidates is an area that accompanies the world trend of development of novel probiotic strains and new products. In recent years, unconventional sources of potential probiotic bacteria have been studied. Furthermore, nowadays there has been a growing interest in non-dairy probiotic products and fermented plant-based foods, which has led to the development of probiotic foods currently being presented as a research priority for the food industry. The aim of this work was to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from Jerusalem artichoke (Helianthus tuberosus L.) tubers. The results proved that the selected isolated LAB strains exhibited a high survival rate in the simulated gastrointestinal treatment, with non-hemolytic nor DNAse activity and antibiotic sensitivity. The isolated strains also showed antimicrobial activity against pathogen microorganisms, due to their acidification capacity. The molecular identification of the bacilli strains showed a high similarity with the genus Lentilactobacillus and, within this genus, with the species kosonis and curieae. Hence, these strains revealed potential probiotic in vitro characteristics that position them to be used in plant-based functional food.