ABSTRACT Probiotics from non-traditional sources (nondairy and non-intestinal) have better potential of surviving in the nondairy based foods that are required to overcome the limitations of dairy-based (traditional) probiotic products. This study aimed to evaluate the probiotic potential of lactic acid bacteria isolated from traditional and non-traditional sources in vitro. Thirteen isolates were evaluated for their probiotic properties, including survival under harsh conditions, antimicrobial activity, cell surface hydrophobicity, auto-aggregation and safety. Nondairy isolates (Limosilactobacillus fermentum AIFe1, Pediococcus pentosaceus BRDb27, Lactiplantibacillus plantarum ACFe58 and Lactiplantibacillus plantarum KCFe63) and dairy isolate (Limosilactobacillus fermentum NCCu21) exhibited excellent beneficial properties. Isolate KCFe63 showed good antioxidant potential (66.91% ± 0.02) and cholesterol assimilation (81.48% ± 0.07), while isolate BRDb27 displayed high adhesion ability (77.98% ± 0.71) toward HT-29 cells whereas isolate ACFe58 revealed antiproliferative effects against MCF-7 cells (40.55% ± 0.15 cell survival). These findings suggest that the non-traditional isolates are promising probiotic candidates and can be further explored for commercialization.