This work presents a comprehensive analytical survey of the antibacterial and antibiofouling properties of sub-100-nanometer scale nanopatterned polymers fabricated using scalable industrial processes that are directly applicable for commercial use. Regular nanopillar arrays of 60 nm height and 60 nm pitch were fabricated on polyethylene terephthalate (PET), polypropylene (PP), acrylic, and nylon polymer films using nanoimprint lithography. Analyses of the influence of material type on antibacterial performance revealed that the nanoscale patterns produced using acrylic and nylon polymers were most suitable as anti-infective nanostructured materials. Further assessment of the influence of the nanopattern geometric parameters on the antibacterial efficacy of acrylic and nylon materials revealed the superlative pattern for inactivating and repelling both Gram-positive and Gram-negative bacteria. Acrylic nanostructured films consisting of nanopillars with a height of 60 nm and pitch of 30 nm, respectively, demonstrated distinctly superior antimicrobial and antibiofouling behavior toward both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacterial species.