Successful implementation of in situ bioremediation is contingent upon understanding how physicochemical and microbial factors affect the formation and dynamics of microbially active regions known as bioactive zones (BAZs). This study demonstrates how a novel fiber optic detection system can be used to test hypotheses concerning real-time, in situ BAZ formation and dynamics. This study focuses on naphthalene transport in saturated porous media containing defined physicochemical and microbial heterogeneities. Biological activity was measured using a lux reporter bacterium, Pseudomonas putida RB1353, that bioluminesces during naphthalene catabolism. Results show that the presence of defined heterogeneities drives the development of BAZs at material-property interfaces where the confluence of naphthalene, dissolved oxygen, and sufficient microbial density is optimal. Thus, despite successful transport of P. putida RB1353 into a sterile low-permeability region containing substrate, BAZ formation in this region was limited by local physicochemical conditions (e.g., naphthalene and dissolved oxygen bioavailability). In another instance, transport of P. putida RB1353 occurred against advective flow, resulting in BAZ formation upgradient of inoculated regions. Defined systems such as this can be used as a basis for predicting localization of activity in complex subsurface systems.