Bacterial colonies that reside on a surface, known as biofilms, are intrinsically impenetrable to traditional antibiotics, ultimately driving research toward an alternative therapeutic approach. Nitric oxide (NO) has gained attention for its biologically beneficial properties, particularly centered around its antibacterial capabilities. NO donors that can release the molecule under physiological conditions (such as S-nitrosothiols) can be utilized in clinical settings to combat bacterial biofilm infections. Herein the authors describe determining a critical concentration of NO necessary to cause >90% reduction of a Pseudomonas aeruginosa biofilm grown on medical grade polyurethane films. The biofilm was grown under optimal culture conditions [in nutrient broth media (NBM) at 37 °C] for 24 h before the addition of the NO donor S-nitrosoglutathione (GSNO) in NBM for an additional 24 h. The cellular viability of the biofilm after the challenge period was tested using varying concentrations of NO to determine the critical amount necessary to cause at least a 90% reduction in bacterial biofilm viability. The critical GSNO concentration was found to be 10 mM, which corresponds to 2.73 mM NO. Time kill experiments were performed on the 24 h biofilm using the critical amount of NO at 4, 8, 12, and 16 h and it was determined that the 90% biofilm viability reduction occurred at 12 h and was sustained for the entire 24 h challenge period. This critical concentration was subsequently tested for total NO release via a nitric oxide analyzer. The total amount of NO released over the 12 h challenge period was found to be 5.97 ± 0.66 × 10(-6) mol NO, which corresponds to 1.49 ± 0.17 μmol NO/ml NBM. This is the first identification of the critical NO concentration needed to elicit this biological response on a medically relevant polymer.