In this review, we present nanofluidic phenomena, particularly as they relate to applications involving analysis of biomolecules within nanofabricated devices. The relevant length scales and physical phenomena that govern biomolecule transport and manipulation within nanofabricated nanofluidic devices are reviewed, the advantages of nanofabricated devices are presented, and relevant applications are cited. Characteristic length scales include the Debye length, the Van der Waals radius, the action distance of hydrogen bonding, the slip length, and macromolecular dimensions. On the basis of the characteristic lengths and related nanofluidic phenomena, a nanofluidic toolbox will be assembled. Nanofluidic phenomena that affect biomolecule behavior within such devices can include ion depletion and enrichment, modified velocity and mobility, permselectivity, steric hindrance, entropy, adsorption, and hydrodynamic interaction. The complex interactions and coupled physics of such phenomena allow for many applications, including biomolecule separation, concentration, reaction/hybridization, sequencing (in the case of DNA) and detection. Examples of devices for such applications will be presented, followed by a discussion of near-term challenges and future thoughts for the field.