Movement is a fundamental characteristic of all living things. This biogenic function is carried out by various nanometer-sized molecular machines. Molecular motor is a typical molecular machinery in which the characteristic features of proteins are integrated; these include enzymatic activity, energy conversion, molecular recognition and self-assembly. These biologically important reactions occur with the association of water molecules that surround the motors. Applied pressures can alter the intermolecular interactions between the motors and water. In this chapter we describe the development of a high-pressure microscope and a new motility assay that enables the visualization of the motility of molecular motors under conditions of high-pressure. Our results demonstrate that applied pressure dynamically changes the motility of molecular motors such as kinesin, F1-ATPase and bacterial flagellar motors.