Antimicrobial resistance (AMR) represents a global health crisis associated with 1.27 million deaths in 2019 [1], [2]. New therapeutic approaches include antimicrobial peptides and proteins (AMPs), which are part of the innate immune defense, as alternatives to antibiotics [3]. Histones represent a family of small basic proteins that associate with DNA, whose antibacterial activity was first reported in 1942. These proteins and their fragments can be found in the cytoplasm, cellular membrane, extracellular fluid, and neutrophil extracellular traps (NETs) [4]. However, the antibacterial activity of human histones or their fragments has never been investigated in detail. We used a hemofiltrate peptide database for antimicrobial peptide prediction to identify novel human AMPs. Thirteen histone H1 sequences were identified as putative AMPs, synthesized, and tested against bacterial ESKAPE pathogens in a radial diffusion assay. A prominent dose-dependent antibacterial activity was detected for twelve fragments of H1 against P. aeruginosa. These histone H1 fragments inhibited the growth of P. aeruginosa without disrupting the bacterial membrane, as shown by a Sytox assay. H1 fragments incubated with THP-1 cells were not cytotoxic in LDH and Alamar assays. The characterized antimicrobial activity points to a role for human histone H1 fragments in innate immunity and may represent a promising approach for the development of novel antibacterial therapies.