Background: Adherence of complex bacterial biofilm communities to burned tissue creates a challenge for treatment, with infection causing 51% of burn victim deaths. This study evaluated the release of therapeutics from wound care biomaterials and their antimicrobial activity against pathogens Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. Methods: Electrospun chitosan membranes (ESCMs) were fabricated and acylated with chain lengths ranging from 6–10 carbons then loaded with 0.15 mg of anti-biofilm agent, cis-2-decenoic acid (C2DA), and 0.5 mg of local anesthetic, bupivacaine. Results: Combinations of therapeutics released from modified ESCMs at a cumulative amount of 45–70% of bupivacaine and less than 20% of C2DA. Results from bacterial studies suggest that this combination reduced biofilm 10-fold for S. aureus, 2-fold for Acinetobacter baumannii, and 2–3-fold for Pseudomonas aeruginosa by 24 hours. Additionally, dual loaded groups reduced planktonic Staphylococcus aureus ~4-fold by 24 hours as well as Acinetobacter baumannii ~3-fold by 48 hours. Conclusions: The combination of therapeutics used has a significant role in biofilm prevention for selected strains via direct contact or diffusion in aqueous solutions.