The patency of small-diameter (<6 mm) synthetic vascular grafts (VGs) is still limited by the absence of a confluent, blood flow-resistant monolayer of endothelial cells (ECs) on the lumen and of vascular smooth muscle cell (VSMC) growth into the media layer. In this research, electrospinning has been combined with bioactive coatings based on chondroitin sulfate (CS) to create scaffolds that possess optimal morphological and bioactive properties for subsequent cell seeding. We fabricated random and aligned electrospun poly(ethylene terephthalate), ePET, mats with small pores (3.2 ± 0.5 or 3.9 ± 0.3 μm) and then investigated the effects of topography and bioactive coatings on EC adhesion, growth, and resistance to shear stress. Bioactive coatings were found to dominate the cell behavior, which enabled creation of a near-confluent EC monolayer that resisted physiological shear-flow conditions. CS is particularly interesting since it prevents platelet adhesion, a key issue to avoid blood clot formation in case of an incomplete EC monolayer or partial cell detachment. Regarding the media layer, circumferentially oriented nanofibers with larger pores (6.3 ± 0.5 μm) allowed growth, survival, and inward penetration of VSMCs, especially when the CS was further coated with tethered, oriented epithelial growth factor (EGF). In summary, the techniques developed here can lead to adequate scaffolds for the luminal and media layers of small-diameter synthetic VGs.