Different types of carbon substrates were widely used in soil remediation. However, differences of their impacts and related mechanisms on degradation of polycyclic aromatic hydrocarbons (PAHs) and microbial community structures in contaminated soil still remain unclear. Here, we investigated the effects of corn straw (S), glucose (G), straw combined with glucose (SG), and sodium azide (N, as an abiotic control) on PAHs fractions and bacterial communities in soil. After 70 days’ microcosm experiments, total PAHs concentrations were significantly reduced by 30.9%, 19.5% and 44.6% under S, G and SG treatments. Water soluble, acid soluble and residual PAHs under all treatments were significantly decreased after 70 days of incubation, while organically bound PAHs were increased by 11.4%, 22.7% and 36.1% under G, S and SG treatments. Additionally, straw and glucose application increased relative abundance related PAHs-degrading bacteria and the copy numbers of gram-negative (PAHs-RHDα GN) and gram-positive genes (PAHs-RHDα GP) in the contaminated soil. Redundancy analysis (RDA) and Random Forest (RF) indicated that PAHs fractions are crucial factors for biodegradation of PAHs in PAHs-contaminated soils amended with carbon substrates. These suggested that carbon substrates contributed to PAHs conversion from residual PAHs (nonlabile fractions) to organically bound PAHs and thus increased the potential for PAHs conversion to water-soluble and organic acid-soluble PAHs, which were more easy to be utilized by soil microorganisms. This study revealed the new insights of different carbon substrates on degradation and dynamic changes of PAHs fractions and the better potential of combined application of straw and glucose in enhancing degradation of PAHs in PAHs-contaminated soils.