Hemoglobin is a known source of biologically active peptides with various functions. In the present study, we report for the first time the existence of natural processed hemoglobin fragments exhibiting antimicrobial activity in humans. Two antimicrobial hemoglobin-derived peptides were purified from a human placental peptide library by consecutive chromatographic steps tracking the maximum growth inhibitory activity against Escherichia coli BL21. These peptides, consisting of 17 and 36 amino acid residues, were identified as being C-terminal fragments of gamma-hemoglobin and beta-hemoglobin, respectively. The antimicrobial beta-hemoglobin fragment was also purified from lysed erythrocytes, demonstrating that proteolytic degradation of hemoglobin into small bioactive peptides already starts inside erythrocytes. The identified peptides inhibit the growth of Gram-positive and Gram-negative bacteria and yeasts in micromolar concentrations. Moreover, by LPS-binding, the beta-hemoglobin fragment reduces biological activity of endotoxins. In contrast, even at high concentrations, the identified antimicrobial hemoglobin peptides do not exhibit toxic activity on human primary blood cells. We conclude that antimicrobial hemoglobin-derived peptides could be important effectors of the innate immune response killing microbial invaders.