Cystic fibrosis (CF) is characterized by chronic infections within the highly viscous mucus layer of the airways. Nitric oxide (NO), an endogenously produced free radical involved in the immune response to foreign pathogens, can be delivered exogenously from macromolecular scaffolds to combat bacterial infection. Herein, the ability of NO-releasing chitosan oligosaccharides to eradicate P. aeruginosa biofilms and alter the biophysical properties of infected airway mucus was characterized. As oxygen gradients form within the CF mucus layer, the antibacterial efficacy of NO was determined in aerobic and anaerobic conditions. Against planktonic cultures, NO-releasing chitosan oligosaccharides were more effective antibacterial agents under anaerobic environments due to reduced NO reactions with oxygen. However, the bactericidal NO dose released from the scaffold was the same under both conditions, indicating that the efficacy of NO against planktonic cultures was independent of oxygen concentration. For P. aeruginosa biofilms, the minimum biofilm eradication concentration of NO-releasing chitosan oligosaccharides was decreased under anaerobic conditions. Furthermore, the concentration of NO required to eradicate biofilms was 10-fold lower than tobramycin under anaerobic conditions. Biofilm disruption and eradication were investigated as a function of NO-releasing chitosan oligosaccharide dose, with results compared to non-NO-releasing chitosan oligosaccharides and tobramycin. Quantification of biofilm expansion/contraction and multiple-particle tracking microrheology were used to assess the biofilm structural integrity before and after antibacterial treatment. While tobramycin had no effect on the physical properties of the biofilm, NO-releasing chitosan oligosaccharides induced biofilm degradation. Control chitosan oligosaccharides actually increased biofilm elasticity, indicating that the scaffold may mitigate the biofilm disrupting power of NO. The effects of NO on the physical properties of CF mucus were examined to assess the mucolytic potential of NO-releasing chitosan oligosaccharides. When released from a mucoadhesive scaffold, NO increased mucin migration during electrophoretic separation, indicating a reduction in mucin molecular weight. Additionally, NO destroyed the physical structure of mucin networks and reduced the viscosity and elasticity of CF sputum. The mucoadhesive properties of each scaffold dictated the mucolytic efficacy of NO-releasing chitosan oligosaccharides, as non-adhesive scaffolds were incapable of altering mucins in purified mucin solutions and CF sputum.