Nanotechnology, the manipulation of matter on atomic, molecular, and supramolecular scales, has become the most appealing strategy for biomedical applications and is of great interest as an approach to preventing microbial risks. In this study, we utilize the antimicrobial performance and the drug-loading ability of novel nanoparticles based on silicon oxide and strontium-substituted hydroxyapatite to develop nanocomposite antimicrobial films based on a poly(l-lactic acid) (PLLA) polymer. We also demonstrate that nanoimprint lithography (NIL), a process adaptable to industrial application, is a feasible fabrication technique to modify the surface of PLLA, to alter its physical properties, and to utilize it for antibacterial applications. Various nanocomposite PLLA films with nanosized (black silicon) and three-dimensional (hierarchical) hybrid domains were fabricated by thermal NIL, and their bactericidal activity against Escherichia coli and Staphylococcus aureus was assessed. Our findings demonstrate that besides hydrophobicity the nanoparticle antibiotic delivery and the surface roughness are essential factors that affect the biofilm formation.