Human amniotic fluid (AF) contains numerous nutrients, trophic factors and defense proteins that provide a nurturing and protective environment for fetal development. Based on reports that AF has antibacterial, anti-inflammatory and regenerative properties, we designed a novel method to process AF for use in clinical care. Six randomly selected lots of processed AF (pAF) were examined to determine whether they retained their antibacterial activity against a panel of wound-associated pathogens E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and E. aerogenes (ESKAPE). To identify proteins in pAF that might be responsible for its antibacterial activity, three different lots of pAF were analyzed with quantitative cytokine arrays that consisted of 400 unique human proteins. One protein identified by microarrays, lactoferrin, and a second prominent antibacterial protein that was not identified by microarrays, lysozyme, were examined by depletion experiments to determine their contribution to the antibacterial activity of pAF. All six lots of pAF exhibited antibacterial activity against ESKAPE microorganisms, especially against the pathogens predominately found in chronic wounds (i.e. S. aureus and P. aeruginosa). Thirty-one of the peptides on the microarray were annotated as having antibacterial activity and 26 of these were detected in pAF. Cystatin C and lactoferrin were among the most highly expressed antibacterial proteins in pAF. Cystatin C and lactoferrin were confirmed by ELISA to be present in pAF along with lysozyme. Immunoprecipitation of lactoferrin and lysozyme reduced, but did not abolish the antibacterial activities of pAF. Our data demonstrate that pAF maintains antibacterial activity via the preservation of antibacterial proteins against a broad spectrum of wound-associated pathogens.