The present study was conducted to explore the mechanisms of surfactant-enhanced phytoremediation of soils contaminated with polycyclic aromatic hydrocarbons (PAHs), focusing on the bioavailability of PAHs and microbial diversity. We investigated the remediation efficiencies of phenanthrene and pyrene after the addition of mixed surfactants (sodium dodecyl benzene sulfonate (SDBS) and Tween 80) of different ratios (1:1, 1:2, and 2:1) at the concentration of 100 mg/kg to soils cultured with ryegrass (Lolium multiflorum L.). The fractions of phenanthrene and pyrene were determined using a sequential extraction method, and the microbial diversity was evaluated using 16S rRNA gene high-throughput sequencing. The results showed that mixed surfactants could enhance the remediation efficiencies of PAHs, and mainly occurred in the initial 21 days. Mixed surfactants at the ratio of 1:1 (HM1) showed the best remediation efficiency in enhancing the dissipation of pyrene in 21 days. Mixed surfactants showed little effects on the removal of phenanthrene. In general, HM1 significantly decreased the bioavailable, bound and residual fractions of pyrene; additionally, higher abundances of PAH-degradation bacteria and degradation-related genes were observed. Pearson correlation analysis among PAH degraders, degradation-related genes and bioavailable fraction of PAHs was performed. Our results indicated that mixed surfactants could promote the transformation of pyrene from the bound and residual fractions to bioavailable fractions and enhance the abundances of PAH degradation bacteria and PAH degradation-related genes, thereby enhancing the degradation of pyrene.