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Biofilms are the cause of major bacteriological infections in patients. The complex architecture of <i>Escherichia coli</i> (<i>E. coli</i>) biofilm attached to the surface of catheters has been studied and found to depend on the biomaterial&#8217;s surface properties. The SEM micrographs and water contact angle analysis have revealed that the nature of the surface affects the growth and extent of <i>E. coli</i> biofilm formation. In vitro studies have revealed that the Gram-negative <i>E. coli</i> adherence to implanted biomaterials takes place in accordance with hydrophobicity, i.e., latex &gt; silicone &gt; polyurethane &gt; stainless steel. Permanent removal of <i>E. coli</i> biofilm requires 50 to 200 times more gentamicin sulfate (G-S) than the minimum inhibitory concentration (MIC) to remove 90% of <i>E. coli</i> biofilm (MBIC₉₀). Here, in vitro eradication of biofilm-associated infection on biomaterials has been done by Eudragit RL100 encapsulated gentamicin sulfate (E-G-S) nanoparticle of range 140 nm. It is 10&#8722;20 times more effective against <i>E. coli</i> biofilm-associated infections eradication than normal unentrapped G-S. Thus, Eudragit RL100 mediated drug delivery system provides a promising way to reduce the cost of treatment with a higher drug therapeutic index.