As more micro- and nanofluidic methodologies are developed for a growing number of diverse applications, it becomes increasingly apparent that the choice of substrate material can have a profound effect on the eventual performance of a device. This is due mostly to the high surface-to-volume ratio that exists within such small structures. In addition to the obvious limitations related to the choice of solvent, operating temperatures, and pressure, the method of fluidic pumping—in particular, an electrokinetics-based methodology using a combination of electro-osmotic and electrophoresis flows—can further complicate material choice. These factors, however, are only part of the problem; once chemicals or biological materials (e.g., proteins or cells) are introduced into a microfluidic system, surface characteristics will have a profound influence on the activity of such components, which will subsequently influence their performance. This article reviews the common types of materials that are currently used to fabricate microfluidic devices and considers how these materials may influence the overall performance associated with chemical and biological processing. Consideration will also be given to the selection of materials and surface modifications that can aid in exploiting the high surface properties to enhance process performance.