Recent advances in multilayer self-assembly have enabled the precise construction of nanocomposite ultrathin films on a variety of substrates, from large-area planar surfaces to nanoparticles. As a result, a wide range of physico-chemical properties may be represented by selecting from an array of surface preparations, molecules, assembly conditions, and post-assembly treatments. Such multilayer nanofilm assemblies are particularly attractive for use as specialized membranes for selective transport, which have many applications for separations, sensors, and drug delivery systems. In this work, nanocomposite ultrathin films built with layer-by-layer (LbL) self-assembly methods have been applied to surface modification to control interfacial behavior, including diffusion, anti-fouling, and biomimetic membranes. Transport and interfacial properties of nanocomposite membranes constructed using LbL self-assembly with synthetic and/or bio-polymers were characterized, and permeability values of clinically relevant small molecules through the nanofilms were determined. Correlations between permeability and film properties were also examined. Nanofilm coatings around 100nm thickness decreased diffusion coefficients of glucose up to five orders of magnitude, and were found to greatly affect enzymatic glucose sensor responses. Surface modification on top of the nanofilms with poly(ethylene glycol) provided anti-fouling effects. However, weak-weak polyelectrolyte multilayers (PEMs) should not be used to control transport due to their susceptibility under normal physiological conditions. Natural/biological polymers also provided multilayer film structures at the specific conditions, but their transport-limiting properties were not significant compared to synthetic PEMs. Even when covalently crosslinked, biological PEMs did not reduce the permeability of a small molecule. Finally, the predicting model of projecting analyte permeation through multi-phase nanocomposite films comprised with known diffusion coefficients was theoretically and experimentally evaluated. The modeling was matched reasonably well to experimental data. The outcomes will be the key knowledge or engineering principles to support future efforts in research and development. It is anticipated that the system developed for determining transport properties will provide a general platform for assessing new candidate materials. The theory developed will be useful in estimating transport properties of novel nanocomposite materials that may be interesting in a broad array of chemical and biological systems, from analytical separations to implantable biomedical applications, and will provide useful design rules for materials and fabrication process selection.