The rising demand for non-dairy probiotic products is driven by factors such as vegetarian diets, concerns about high cholesterol in milk and lactose intolerance. This research investigated the presence of Lactobacillus plantarum in apple, pear, and quince vinegar using molecular and biochemical methods. Isolated microorganisms were evaluated for probiotic potential based on their ability to grow at different bile salt concentrations and pH levels. Biochemical characterization included sugar fermentation profile, presence of extracellular enzymes and antibiotic susceptibility testing. Molecular identification of strains employed specific L. plantarum recA (Recombinase A) primer targeting the recA gene, which encodes a multifunctional protein essential for bacterial cells. Among the 24 microorganisms isolated from apple, pear, and quince vinegar, nine strains displayed a specific band with the L. plantarum recA primers, confirming their identity. These Gram-positive bacteria were positive for lipase and protease activity but negative for catalase, amylase, gelatinase, and oxidase. The L. plantarum strains fermented all tested sugars except xylose and demonstrated tolerance to acidic and bile-containing environments, high temperatures, and salt concentrations. While resistant to eight of the 15 antibiotics tested, the bacteria showed relative sensitivity to three. Furthermore, they exhibited anti-proliferative effects on human HT-29 cancer cells, suggesting potential as anticancer agents. This study successfully isolated L. plantarum strains from apple, pear, and quince vinegar with promising probiotic and anticancer properties.