This study was undertaken to assess the antibacterial efficacy of lactobacilli isolated from curd and human milk samples. Identities of thirty-one different lactobacilli (20 from curd and 11 from human milk) were confirmed by genus-specific PCR and 16S rRNA-based sequencing. These strains belonged to five species, Lactobacillus casei, L. delbrueckii, L. fermentum, L. plantarum, and L. pentosus. Antibacterial activities of cell-free supernatants (CFSs) of all the Lactobacillus isolates were estimated through standard agar-well diffusion assay, against commonly occurring food-borne and clinically important human pathogens. None of the lactobacilli cell-free supernatant (CFS) exhibited inhibitory activity against four pathogens, namely Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Klebsiella pneumoniae. Bacillus cereus, Salmonella enterica serovar Typhi, and Shigella flexneri were moderately inhibited by majority of CFSs, whereas, weak activity was observed against Pseudomonas aeruginosa and Proteus mirabilis. CFS of some of the curd isolates displayed antagonistic activity against Streptococcus mutans; however, human milk lactobacilli did not displayed any inhibitory activity against them. As expected, Nisin (Nisaplin®) showed inhibitory activity against Gram-positive, S. aureus, B. cereus, and L. monocytogenes. Interestingly, few of the examined CFSs exhibited inhibitory activities against both Gram-positive and Gram-negative pathogens. Findings from this study support the possibility to explore the tested lactobacilli and their CFSs as natural bio-preservatives, alone or in combination with approved bacteriocins in food and pharma formulations after validating their safety.