In recent years, the demand for the use of natural antimicrobial agents rather than chemicals as food preservatives is rapidly growing. The whole bacterial culture and neutralized supernatants of thirty-nine isolates were examined for antimicrobial activity by the spot-on lawn assay against ten indicator microorganisms. Based on the results, the cultures of all isolates showed antimicrobial activity. The maximum inhibition zone diameter was 38.2±0.1 mm. All the isolates had antimicrobial effects against Bacillus cereus, while 88.88% of the isolates did not show any inhibition zone around Candida albicans. Finally, of eleven selected broad spectrum isolates; only isolate DL200 had antimicrobial activity even in a neutralized supernatant. The range of lactic acid production was 14.2-34.8 mg/g in cultured skim milk. There is a correlation coefficient between lactic acid production and the antimicrobial activity of isolates only against Staphylococcus aureus and Bacillus subtilis. However, there is no correlation between the lactic acid production of isolates and antimicrobial activity against other indicators, like Salmonella typhimurium, Escherichia coli, and Pseudomonas aeruginosa, and increasing lactic acid production did not increase antimicrobial activity. Selected isolates which use galactose as the sole carbon source for the production of CO₂, were characterized as facultative heterofermentative and were identified as Lactobacillus paracasei with 99-100% identity based on the alignment results of their 16S rRNA gene. The use of selected isolates as preservatives in food not only inhibits the growth of pathogenic microorganisms but also prevents D-lactic acidosis in the host. Therefore, they are essential for children in the Autism spectrum who have high D-lactate in their system.