Owing to the probiotic origin, lipases-derived from the Lactobacilli sp. are considered to be promising biomaterials for in vivo applications. On a different note, poly(ε-caprolactone) (PCL)—an FDA-approved polymer for implantable applications—lacks inherent antimicrobial property, because of which suitable modifications are required to render it with bactericidal activity. Here, we employ Lactobacillus amylovorous derived lipase to surface derivatize the PCL films with silver that is a highly efficient inorganic broad-spectrum antimicrobial substance. Two different surface functionalization strategies have been employed over the alkaline hydrolyzed PCL films towards this purpose: In the first strategy, lipase-capped silver nanoparticles (Ag NPs) have been synthesized in a first step, which have been covalently immobilized over the activated carboxylic groups on the PCL film surface in a subsequent step. In the second strategy, the lipase was covalently immobilized over the activated carboxylic groups of the PCL film surface in the first step, over which silver was deposited in the second step using the dip-coating method. While the characterization study using X-ray photoelectron spectroscopy (XPS) has revealed the successful derivatization of silver over the PCL film, the surface characterization using field-emission scanning electron microscopy (FE-SEM) study has shown a distinct morphological change with higher silver loading in both strategies. The antimicrobial studies employing E. coli have revealed 100 % inhibition in the bacterial growth in 4–6 h with the Ag NPs-immobilized PCL films as opposed to >8 h with those prepared through the dip-coating method. Additionally, the cytotoxicity assay using mouse fibroblast cells has shown that the PCL films immobilized with lipase-capped Ag NPs exhibit high cell compatibility, similar to that of pristine PCL film, and thereby making it suitable for in vivo applications.