This study focused on screening soil bacteria that possess antibacterial properties, sourced from various locations in Hisar, Haryana, India. To address this, the researchers employed standard techniques to isolate and characterize soil bacteria exhibiting antibacterial activity. The bacteria were tested against harmful human pathogens such as Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa by using primary and secondary screening methods. Isolates SB1, SB2, and SB3 demonstrated antibacterial properties against all three pathogens in both screening methods. The effects of filter sterilization, autoclaving, and proteinase K treatment were studied on the culture filtrates. 16S rRNA sequencing identified the isolates SB1, SB2, and SB3 as Neomicrococcus lactis strain PR-F11, Nocardia Alba strain YIM 30243 and Bacillus cereus NC7401, respectively. Out of 208 soil samples, only 3 bacterial isolates displayed antibacterial activity against human pathogens S. aureus, E. coli, and P. aeruginosa. The isolates, Neomicrococcus lactis strain PR-F11, Nocardia Alba strain YIM 30243 and Bacillus cereus NC7401, were identified as Gram-positive by 16S rRNA studies. Culture filtrates of these isolates were tested for efficacy A comparison of the antibacterial activity of culture filtrates and antibiotic streptomycin showed that culture filtrates produced an inhibition zone of 19.5 mm. These 3 bacterial strains, showed antibacterial activity against all the mentioned human pathogens. The antibiotic factor was found to be proteinaceous, as proteinase K significantly reduced the antibacterial activity of the culture filtrates. Based on these significant results, these 3 bacteria are promising candidates for the isolation of new antibacterial agents.