Histone‐derived antimicrobial peptides have been identified in various organisms from plants to humans. The rat histone H4 mRNA variants, H4‐v.1 and rat histogranin (HNr) mRNAs, were recently reported to be involved in the synthesis of H4‐(86–100) and its related peptide HNr, respectively. Herein, the two peptides were investigated for putative antimicrobial activity and found to inhibit growth of Gram‐negative (Escherichia coli, Pseudomonas aeruginosa) and Gram‐positive (Bacillus subtilis, Staphylococcus aureus) bacteria. Their inhibitory potencies in E. coli (LD50: 3.48 and 4.34 μg·mL−1) are comparable to that of the antimicrobial peptide LL‐37 (LD50: 4.10 μg·mL−1). The antimicrobial activities of H4‐(86–100) and HNr depend upon the integrity of the molecules, as precursors [H4‐(84–102), pro‐HNr] and fragments [bovine histogranin (HNb)‐(1–13), HNb‐(3–13), H4‐(89–102) or OGP] are at least five times less potent than the parent peptides. Among various HN‐like compounds, cyclo‐(‐Gly‐pCl‐Phe‐Tyr‐d‐Arg) (compound 3) and N‐5‐guanidino pentanamide‐(2R)‐yl‐2‐N‐(p‐hydroxyphenylacetyl)‐4‐(p‐chlorobenzoyl)‐phenylene diamine (compound 8) display antimicrobial activities comparable to that of HNr. Interestingly, the antimicrobial activities of H4‐(86–100), HNr and compound 3, like those of quinolone antibiotics acting as DNA gyrase poisons, are potentiated by ATP (1 mm) and coumermycin A1 (a DNA gyrase‐linked ATPase inhibitor) and blocked by 2,4‐dinitrophenol (DNP, an uncoupler of oxidative phosphorylation) and fluoroacetic acid (a metabolic poison). Finally, in vitro experiments indicate that H4‐(86–100), HNr, compound 3 and compound 8, but not HNb‐(1–13) or HNb‐(3–13), inhibit DNA gyrase‐mediated supercoiling of pBR322 DNA. These data indicate that the naturally occurring H4‐(86–100) and HNr display antimicrobial effects that involve a modulation of ATP‐dependent DNA gyrase.