The use of medical devices has facilitated the management of serious illnesses. However, the insertion of foreign material into the body creates a favorable niche for bacterial adhesion and biofilm formation. Biofilm formations on medical devices have contributed significantly to increased patient morbidity and mortality. Biofilm infections are often chronic and recalcitrant to antibiotic therapy. Biofilms of bacteria are composed of polysaccharides, proteins, and extracellular DNA (eDNA). eDNA is an integral part of the biofilm and plays a vital role in maintaining the 3D architecture of the biofilm. We investigated the effect of ceftazidime (3rd generation cephalosporin), gentamicin (aminoglycoside) and norfloxacin (fluoroquinolone) antibiotics on biofilm formation and eDNA release in ATCC 27853 strain and clinical isolates of Pseudomonas. We observed that all clinical isolates were resistant to ceftazidime. Our study showed that ceftazidime, gentamicin and norfloxacin at sub-inhibitory concentrations induced biofilm formation. Half MIC concentrations increased biofilm formation by both ATCC 27853 strain and clinical isolates. On the other hand, an increase in eDNA release was observed only at sub-inhibitory concentrations of gentamicin and norfloxacin, observation of clinical relevance, as bacteria are continuously exposed to sub-lethal concentrations of antibiotics. An increase in biofilm formation and eDNA release at sub-inhibitory concentrations of antibiotics suggest a protective mechanism by bacteria to resist antibiotics and persist within the host and indwelling medical devices.