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We successfully produced two human β-defensins (hBD-1 and hBD-2) in bacteria as functional peptides and tested their antibacterial activities againstSalmonella enterica serovar Typhi,Escherichia coli , andStaphylococcus aureus employing both spectroscopic and viable CFU count methods. Purified peptides showed approximately 50% inhibition of the bacterial population when used individually and up to 90% when used in combination. The 50% lethal doses (LD50 ) of hBD-1 againstS. Typhi,E. coli , andS. aureus were 0.36, 0.40, and 0.69 μg/μl, respectively, while those for hBD-2 against the same bacteria were 0.38, 0.36, and 0.66 μg/μl, respectively. Moreover, we observed that bacterium-derived antimicrobial peptides were also effective in increasing survival time and decreasing bacterial loads in the peritoneal fluid, liver, and spleen of a mouse intraperitoneally infected withS. Typhi. The 1:1 hBD-1/hBD-2 combination showed maximum effectiveness in challenging theSalmonella infectionin vitro andin vivo . We also observed less tissue damage and sepsis formation in the livers of infected mice after treatment with hBD-1 and hBD-2 peptides individually or in combination. Based on these findings, we conclude that bacterium-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.