Cystic fibrosis (CF), an atypical genetic disorder, develops due to mutations in cystic fibrosis transmembrane conductance regulator gene, which consequently leads to infection and inflammation. CF infections are commonly characterized by the presence of an extracellular polymeric substance (EPS) matrix or the ‘biofilm’, which presents an entry barrier for the antibiotics. The current research work focuses on systematic Quality by Design based development of cefoperazone sodium loaded liposome formulation. DPPC and cholesterol containing liposomes were formulated by using ‘thin film hydration’ method. The freeze drying and further characterization of optimized formulation was carried out for particle size distribution, % entrapment efficiency, FTIR, DSC and pXRD. The IC₅₀ value of the formulation (0.42 μg/ml) was found to be half of that of the drug (0.92 μg/ml). The formulation showed 50% biofilm inhibition and eradication at ~1 μg/ml. The cell surface hydrophobicity was reduced to ~50% at MIC value of the formulation while it was 78% for the control. The EPS component of P. aeruginosa biofilm reduced to 17% after treatment with 0.42 μg/ml formulation. The effect of formulation on biofilm was further confirmed by SEM analysis which revealed that the biofilm was disintegrated on treatment with 0.42 μg/ml of formulation.