The fabrication of micron-scale channels and reaction chambers using micromachining techniques has created devices with large surface to volume ratios. As a result, surface properties play a major role in determining the behavior of micromachined devices. In this work, we present strategies that can be used to reconfigure surfaces from hydrophobic to hydrophilic or from hydrophilic to hydrophobic. The reversible nature of the surface is made possible by using deposition and removal of biomolecules or amphiphiles on self-assembled monolayers (SAMs). When the initial surface was hydrophobic (using a CH3-terminated SAM on the surface, water contact angle ∼100), it was rendered hydrophilic (water contact angle ≤60°) using monolayer adsorption of avidin protein. To retrieve the hydrophobicity, the avidin was subsequently removed using non-ionic surfactant octyl-β-D-glucopyranoside. Moreover, by incorporating a biotinylated poly(ethyleneglycol), the avidin-coated surface was resistant to further non-specific adsorption. If the initial surface was hydrophilic (using a CO2H-terminated SAM on the surface, water contact angle ≤20°), it was rendered hydrophobic (water contact angle >90°) using monolayer amphiphilic octadecylamine adsorption. The hydrophilicity was restored after subsequently removing the amphiphile using anionic surfactant sodium lauryl sulfate. Both types of surfaces showed excellent reversibility and demonstrated the ability to control surface wettability.