Crude oil is a serious soil pollutant, requiring large-scale remediation efforts. Bacterial consortia in combination with rhamnolipids can be an effective bioremediation method. However, the underlying mechanisms and associated changes in soil bacterial composition remain uncharacterized. Therefore, this study sought to evaluate the effectiveness of rhamnolipids in petroleum hydrocarbon removal, and the associated bacterial community dynamics during bioremediation of petroleum-contaminated soils. Contaminated soils were subjected to natural attenuation, bioremediation with rhamnolipids, bioremediation with bacterial consortia, or bioremediation with bacterial consortia supplemented with rhamnolipids (BMR). High-throughput sequencing of bacterial sample partial 16S rRNA sequences was performed. Additionally, the n-alkanes and aromatic fractions were analyzed by gas chromatography-mass spectroscopy. The results showed that rhamnolipid supplementation increased the rate and extent of total petroleum hydrocarbon biodegradation to a maximum of 81% within 35 days. Further, phylogenetic analysis revealed that the bacterial community was composed of 14 phylotypes (similarity level = 97%). Actinobacteria and Proteobacteria were the two core phyla in all samples, accounting for 63-89%, but Proteobacteria was the most dominant phylum in the BMR sample (similar to 53%). Among the top 20 genera, Pseudomonas, Pseudoxanthomonas, Cavicella, Mycobacterium, Rhizobium, and Acinetobacter were more abundant in BMR samples compared to other samples. Predicted functional profiles revealed that rhamnolipid addition also induced changes in gene abundance related to hydrocarbon metabolic pathways. This study provided comprehensive insights into the synergistic effect of rhamnolipids and bacterial consortia for altering bacterial populations and specific functional traits, which may serve to improve bacteria-mediated petroleum hydrocarbon biodegradation in contaminated soils.