The study of nanomaterial translocation across epithelial barriers is often hindered by the low permeability of transwell membranes to nanoparticles. To address this issue ultra-thin poly(L-lactic acid) nanofilms with zero tortuosity micropores were developed for use in nanoparticle passage tests. In this study we demonstrate that microporous polymeric nanofilms allow a significantly higher passage of silver nanoparticles in comparison with commercial membranes normally used in transwell inserts. A reliable procedure for collecting free-standing nanofilms which enables their manipulation and use in lab-on-chip systems is described. We also demonstrate the cytocompatibility of porous nanofilms and their ability to sustain the adhesion and proliferation of Caco-2 cells. Ultra-thin microporous membranes show promise as low-cost nanomaterial screening tools and may be used as matrices for the development of bioengineered systems for mimicking the intestinal epithelium.