Traditionally, solvent extractions are routinely used in the assessment of contaminated land. However, vigorous solvent extractions only give total concentrations rather than that relating to the bioaccessible fraction. Recently, less harsh, aqueous-based extraction methods have been shown to be a better estimate of the microbial degradation of polycyclic aromatic hydrocarbons (PAHs). The aqueous-based hydroxypropyl-β-cyclodextrin (HPCD) extraction technique was tested using 14C-PAHs in soils and compared against indigenous microbial mineralization (a measure of bioaccessibility) of 14C-phenanthrene in the presence of pyrene or benzo[a]pyrene (B[a]P) over a range of concentrations (0, 5, 10, or 50 mg kg−1) and aged for 0, 25, 50, and 100 d in four soils. At each time point, the total loss, extractability, and mineralization of 14C-phenanthrene was measured in each of the soils. The presence of the other PAHs had little effect on the behavior of 14C-phenanthrene in any of the soils. Comparisons between the amounts of 14C-phenanthrene extracted using HPCD and mineralized were made and showed that there was a correlation (1:1). This study demonstrates that HPCD extraction is able to predict the microbial accessibility fraction of 14C-phenanthrene in the presence of other PAHs in a range of soils, further supporting the applicability of this technique.