The role of humic acid (HA) in the biodegradation of toxic polycyclic aromatic hydrocarbons (PAHs) has been the subject of controversy, particularly in unsaturated environments. By utilizing an infrared spectromicroscope and a very bright, nondestructive synchrotron photon source, we monitored in situ and, over time, the influence of HA on the progression of degradation of pyrene (a model PAH) by a bacterial colony on a magnetite surface. Our results indicate that HA dramatically shortens the onset time for PAH biodegradation from 168 to 2 h. In the absence of HA, it takes the bacteria about 168 h to produce sufficient glycolipids to solubilize pyrene and make it bioavailable for biodegradation. These results will have large implications for the bioremediation of contaminated soils.