Lactic acid bacteria (LAB) have an effective antimicrobial effect against pathogenic bacteria linked to food-borne diseases, including E.coli, Salmonella, Listeria, and other human pathogenic bacteria. LAB produces antimicrobial substances, primarily lactic acid, which lowers pH and creates an acidic environment that inhibits the growth of harmful bacteria. Identified LAB strains, predominantly Lactobacillus species, exhibited probiotic potential and effectively inhibited various tested pathogens. The study collected twelve samples from natural dairy products, homemade pickles, and infant faeces. Catalase assays confirmed the isolates were catalase-negative. The findings revealed that raw milk contains a diverse microbial community rich in LAB, which possesses antagonistic properties. After the Antimicrobial assay of LAB species, the best results were LA2, LA4, LA5, LA7, and LA12. The most effective results after Optimization tests were LA2 and LA7 whose sources are Breast Milk and Infant faeces. Other LAB isolates that tested positive for catalase activity were identified as Bacillus species, exhibiting antifungal properties against different pathogenic fungi such as Aspergillus, Trichoderma, Fusarium, and Rhizoctonia. After fungal assay of Bacillus species, the best isolates were B1, B4, and B5. According to these results, we conducted Optimization tests. Selected isolates B1 and B4 displayed stability across different pH levels, temperatures, and incubation periods, with B4 giving more inhibition zone. Bacillus species produced soluble antibiotics, highlighting their potential in medical and pharmaceutical applications. Overall, LAB and Bacillus isolates present promising opportunities for biological control of diseases in humans, animals, and plants, suggesting their use as effective biological control agents.