We successfully produced two human β-defensins (hBD-1 and hBD-2) in bacteria as functional peptides and tested their antibacterial activities against Salmonella typhi, Escherichia coli and Staphylococcus aureus employing both spectroscopic and viable CFU count methods. Purified peptides showed approximately 50% inhibition of bacterial population when used individually and up to 90% when used in combinations. LD50 values of hBD-1 against S. typhi, E. coli and S. aureus were evaluated as 0.36, 0.40 and 0.69 μg/μl, respectively; while that for hBD-2 against above bacteria were 0.38, 0.36 and 0.66 μg/μl, respectively. Moreover, we observed bacteria derived antimicrobial peptides were also effective in increasing the survival time and decreasing the bacterial load in peritoneal fluid, liver and spleen of a mouse intraperitoneally infected with S. typhi. The 1:1 (hBD-1: hBD-2) combination showed maximum effectiveness in challenging the Salmonella-infection both in vitro and in vivo. Alongside, we observed less tissue damage and sepsis formation in liver of infected mice after treatment with hBD-1 and hBD-2 peptides individually or in combinations. Collectively, based on these findings, we conclude that bacteria-derived recombinant β-defensins (hBD-1 and hBD-2) are promising antimicrobial peptide (AMP) based substances for the development of new therapeutics against typhoid fever.