Probiotics are live cells with various beneficial properties that have been thoroughly researched and investigated for use in a wide range of products on the global market. Numerous scientific researchers have demonstrated their benefits for both human and animal health. The current study set out to isolate probiotic bacteria that could be hostile from a variety of curd samples in order to isolate them. After a preliminary screening process, 39 bacterial strains were identified as promising probiotics from the samples. The probiotic qualities and antagonistic activity of each of the chosen isolates against clinical stool samples obtained from patients and utilized for the isolation of bacterial pathogens were then assessed in vitro. Pathogens and aggregation tests using automated identification systems (VITEK) were used to identify the pure bacterial isolates. The results demonstrated that the most efficient strains for preventing the growth of all test pathogens, including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Proteous vulgaris, and Enterobacter sp., were the prospective probiotic isolates Lact, S2, M3, F1, Y1, Y3, and Y4. The isolates were identified as excellent, promising in vitro antibacterial probiotic isolates against pathogens based on the data obtained; further in vivo evaluation and human health benefits in their actual environments are required.