Bioremediation offers a sustainable approach for removal of polycyclic aromatic hydrocarbons (PAHs) from the environment; however, information regarding the microbial communities involved remains limited. In this study, microbial community dynamics and the abundance of the key gene (PAH-RHDα) encoding a ring hydroxylating dioxygenase involved in PAH degradation were examined during degradation of phenanthrene in a podzolic soil from the site of a former timber treatment facility. The 10,000-fold greater abundance of this gene associated with Gram-positive bacteria found in phenanthrene-amended soil compared to unamended soil indicated the likely role of Gram-positive bacteria in PAH degradation. In contrast, the abundance of the Gram-negative PAHs-RHDα gene was very low throughout the experiment. While phenanthrene induced increases in the abundance of a small number of OTUs from the Actinomycetales and Sphingomonadale, most of the remainder of the community remained stable. A single unclassified OTU from the Micrococcaceae family increased ~20-fold in relative abundance, reaching 32% of the total sequences in amended microcosms on day 7 of the experiment. The relative abundance of this same OTU increased 4.5-fold in unamended soils, and a similar pattern was observed for the second most abundant PAH-responsive OTU, classified into the Sphingomonas genus. Furthermore, the relative abundance of both of these OTUs decreased substantially between days 7 and 17 in the phenanthrene-amended and control microcosms. This suggests that their opportunistic phenotype, in addition to likely PAH-degrading ability, was determinant in the vigorous growth of dominant PAH-responsive OTUs following phenanthrene amendment. This study provides new information on the temporal response of soil microbial communities to the presence and degradation of a significant environmental pollutant, and as such has the potential to inform the design of PAH bioremediation protocols.