Although there are reports on the inhibition of anthropogenic organic chemicals biodegradation due to binding to dissolved humic substances (HS), there is an increasing body of evidence pointing to an enhancing effect in the case of hydrophobic chemicals, like pyrene. The addition of humic fractions to contaminated soils often causes an enhanced biodegradation and desorption of these compounds from soils. Other mechanisms proposed as operating in HS-mediated enhancements of biodegradation include the promotion of compound solubility and a direct access to HS-sorbed chemicals due to the physical association of bacteria and HS. Here, we propose the use of partitioning techniques using poly(dimethylsiloxane) (PDMS) to study the effect of binding of pyrene to a dissolved humic acid isolated from soil on biodegradation of this PAH by a representative soil bacterium. The application of these techniques in biodegradation studies may solve many questions about enhancements in diffusive mass transfer, in capacity/speciation and in dissolution. Therefore, our study may provide new insights into the effects of HS on microbial degradation of polycyclic aromatic hydrocarbons (PAHs).