Aims To assess the biodegradation potential of mixed polycyclic aromatic hydrocarbons (PAHs) in mangrove sediments. Methods and Results Sediment microcosms were constructed with sediment collected from Don Hoi Lot, Samut Songkram Province, Thailand, by supplementation with a mixture of acenaphthene, phenanthrene and pyrene. At the end of 8weeks, low molecular weight PAHs, acenaphthene and phenanthrene were completely degraded. PCR-denaturing gradient gel electrophoresis profile suggests that Marinobacter, Enterobacter and Dethiosulfatibacter play important roles in PAH degradation in mangrove sediment. Furthermore, six PAH-degrading bacteria were isolated consisting of novel phenanthrene-degrading Dyella sp. and Luteibacter sp., phenanthrene-degrading Burkholderia sp., acenaphthene-degrading Alcaligenes sp. and pyrene-degrading Ochrobactrum sp. Moreover, dioxygenase genes could be detected both in sediment microcosms as well as in all of the isolated strains. Conclusions These results demonstrated that indigenous bacteria in the mangrove sediment had the ability to degrade phenanthrene in the presence of mixture PAHs with high efficacy. Significance and Impact of the Study Culture and nonculture methods were combined to assess PAH biodegradation in mangrove sediment. Two novel phenanthrene-degrading bacteria were isolated. Three genera of bacteria that play important roles in PAH degradation were indicated by nonculture approach. Moreover, dioxygenase genes could be detected. This information is useful for further bioremediation of PAH-contaminated mangrove sediments.