The use of renewable, bio-based fuels has become increasingly widespread in recent years, with a major example being biodiesel, a bio-derived alternative to Number 2 diesel fuel. The increased usage of biodiesel gives rise to an augmented need to understand its tribological effects on critical engine components. This study focused on determining the tribological performance of soybean-based B100 (i.e., pure) biodiesel within a fuel injector with varying oscillating frequency by performing a series of linear reciprocating tribological tests of biodiesel-lubricated interfaces with varying reciprocating frequency. Comparison of friction coefficient variation with reciprocating frequency indicated a transition from boundary lubrication to hydrodynamic lubrication as the frequency increased, while hysteresis loop and energy loss observations showed a transition between full stick and partial slip contact with increasing frequency. However, observations of induced wear showed the wear to increase with increasing frequency, most likely due to the augmented number of sliding cycles as well as an increased degree of interfacial slip.