The application of surfactants for the bioremediation of sites contaminated with polycyclic aromatic hydrocarbons has been widely reported, because they are known to increase PAH solubility and desorption, thereby enhancing their bioavailability to biofilm microorganisms. The effects of a nonionic surfactant on the biodegradation of PAHs in porous media, as well as the fate of the surfactant, were investigated in this study. Column experiments in the presence of the surfactant showed that the degradation of the two-ring PAH alone was not significantly affected, but that there was a small enhancement of three- and four-ring PAH degradation when they were present as sole substrates and when using Triton X-100. This was due to the higher solubility of the PAHs in the presence of the surfactant. Biofilm seemed to respond well to binary mixtures of phenanthrene–naphthalene and pyrene–naphthalene, with removals of 45.5 and 24.1%, respectively, in the presence of the surfactant; however, higher biodegradations were always achieved by having just PAH mixtures without the surfactant, indicating the importance of cometabolic mechanisms over improved solubilization of PAHs. Optical sections taken using a confocal laser scanning microscope allowed observation of a heterogeneous web-like matrix of biofilm, with diverse biological aggregate structures.