The polycyclic aromatic hydrocarbons (PAHs) that enter the aqueous phase usually coexist with fulvic acid (FA). Therefore, we initiated this investigation to explore the influences of FA on bacterial biofilm formation and its potential to biodegrade pyrene (PYR), using electron microscopic techniques and isobaric tags for relative and absolute quantification (iTRAQ). Our results revealed that FA stimulated biofilm formation and enhanced the biodegradation of PYR. First, FA favored the three-dimensional proliferation of bacteria, with an OD590/OD600 value of up to 14.78, and the extracellular surfaces covered by a layer of biomaterials. Distinctive intracellular morphologies of texture and organization were accompanied by reduced inter-bacterial distances of less than 0.31 μm. The biofilms formed displayed interactions between FA and surficial proteins, as noted by band shifts for the C–O and CO groups. Strikingly, FA triggered the upregulation of 130 proteins that were either operational in biofilm formation or in metabolic adjustments; with the changes supported by the increasing intensity of free amino acids and the newly generated N–O bonds. The results above revealed that the enhanced biodegradation was related to the up-regulation of the proteins functioned for ribosomal and carbon metabolism, and the ultra-structural changes in FA-induced biofilm system.