Staphylococcus aureus, a human pathogen associated with many illnesses and post-surgical infections, can resist treatment due to the emergence of antibiotic-resistant strains and through biofilm formation. The current treatments for chronic biofilm infections are antibiotics and/or surgical removal of the contaminated medical device. Due to higher morbidity and mortality rates associated with overuse/misuse of antibiotics, alternate treatments are essential. This study reports the antibiofilm activity of avian erythrocyte histones against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). Fluorescence and scanning electron microscopy revealed membrane damage to bacteria in histone-treated biofilms. Histones and indolicidin (positive control) increased the expression of apsS and apsR, which are associated with the Antimicrobial Peptide (AMP) sensor/regulator system in S. aureus. The expression of dltB, and vraF, associated with AMP resistance mechanisms, were under histone inducible control in the biofilm-embedded bacterial cells. The time kill kinetics for histones against S. aureus revealed a rapid biocidal activity (<5 min). Purified erythrocyte-specific histone H5 possessed 3-4 fold enhanced antimicrobial activity against planktonic cells compared to the histone mixture (H1, H2A, H2B, H3, H4, H5). These results demonstrate the promise of histones and histone-like derivatives as novel antibiotics against pathogens in their planktonic and biofilm forms.