In the present study, positive matrix factorization (PMF) and compound-specific isotope analysis were used to investigate the in situ biodegradation of polybrominated diphenyl ethers (PBDEs) in sediment cores collected from a pond at an e-waste recycling site in South China. The potential microorganisms relevant to the degradation of PBDEs were also assessed to aid in the understanding of in situ biodegradation. The PMF results suggested that reductive debromination took place in the sediments. The debromination signal (ratio of the concentration of factor 5 (PMF result) to the total PBDE content) was positively correlated with the relative abundance of Dehalococcoidetes at different core depths. The clear 13C enrichment of five PBDE congeners (BDE 28, 47, 49, 99, and 153) with increasing core depth indicated that a measurable change in isotope fractionation might have occurred during PBDE biodegradation. The in situ biodegradation was further validated by the widespread detection of mono-BDE congeners (BDE 2, BDE 3) and diphenyl ether in the sediments. This study provides new evidence to enhance our understanding of the in situ biodegradation of PBDEs and suggests that the extensive removal of bromine from PBDEs was mediated by indigenous microorganisms at the e-waste site.