Biofilm infections are of great clinical importance due to their high tolerance to antibiotics. Hence, they significantly contribute to the increasing infectious disease-associated mortality. For instance, chronic wounds and cystic fibrosis (CF) lung infections, which are good examples of biofilm-associated infections, cost the UK NHS more than £5Bn yearly. Hence, the need for effective treatments against these infections. We identified a complex medieval infection remedy – “Bald’s Eyesalve” – with good antibacterial efficacy. We reconstructed this complex remedy and redefined it into a simpler and more defined formulation (called Baldexin). We then explored its mechanism of action and resistance evolution pattern. Here, we report that Baldexin has enhanced activity against biofilm-associated populations of Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa in infection-mimicking models, compared with the original remedy. This antibiofilm formulation caused more than 4-log10 killing of S. aureus (methicillin-susceptible and resistant strain), A. baumannii, and P. aeruginosa in an in vitro soft tissue biofilm model. Additionally, we observed a complete eradication of P. aeruginosa biofilms in an ex vivo cystic fibrosis lung model. We also report that “Baldexin” significantly disrupts bacterial membrane integrity and inhibit the expression of genes involved in the de novo purine and pyrimidine biosynthesis pathway. Lastly, both S. aureus and P. aeruginosa showed reduced resistance evolution to this antimicrobial formulation compared to mainline antibiotics. We have shown that this natural product cocktail could potentially be developed into a good treatment for chronic bacterial biofilm infections, including chronic wounds and CF lung infections.