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PAH biodegradation aided by surfactants and cyclodextrins has been widely studied because the former's micelles and the latter's hydrophobic cavities can increase the apparent solubility of PAHs. In this work, the effects of Triton X-100 (TX-100) and β-cyclodextrin (β-CD) on the bioavailability and biodegradation of phenanthrene (PHE) were investigated. The degrading microorganism used was Moraxella osloensis CFP312, a new strain screened from polluted soil and could form biofilms on crystalline PHE. At the same concentrations, β-CD solubilized less PHE than TX-100 but promoted PHE biodegradation. The two solubilizers had almost no different effects on CFP312 hydrophobicity; however, β-CD significantly eluted fewer cells from biofilms on crystalline PHE than TX-100. Microscopy observation revealed that the residual structure of the biofilm treated with β-CD was more complete than that of the biofilm treated with TX-100. This effect is beneficial to the function of biofilms and conducive to mass transfer from the exposed area in the crystalline PHE to the aqueous phase. Although TX-100 and β-CD could enhance the mass transfer of PHE to the aqueous phase, the latter significantly improved the bioavailability and biodegradation of PHE mainly because its solubilization was accompanied by easy desorption and retention of the biofilm functional integrity.