To identify and obtain the indigenous degraders metabolizing phenanthrene (PHE) and biphenyl (BP) from the complex microbial community within industrial wastewater, DNA-based stable-isotope probing (DNA-SIP) and cultivation-based methods were applied in the present study. DNA-SIP results showed that two bacterial taxa (Vogesella and Alicyclobacillus) were considered the key biodegraders responsible for PHE biodegradation only, whereas Bacillus and Cupriavidus were involved in BP degradation. Vogesella and Alicyclobacillus have not been linked with PHE degradation previously. Additionally, DNA-SIP helped reveal the taxonomic identity of Ralstonia-like degraders involved in both PHE and BP degradation. To target the separation of functional Ralstonia-like degraders from the wastewater, we modified the traditional cultivation medium and culture conditions. Finally, an indigenous PHE- and BP-degrading strain, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by enrichment of the 16S rRNA gene and distinctive dioxygenase genes in the DNA-SIP experiment. Our study has successfully established a program for the application of DNA-SIP in the isolation of the active functional degraders from an environment. It also deepens our insight into the diversity of indigenous PHE- and BP-degrading communities.IMPORTANCE The comprehensive treatment of wastewater in industrial parks suffers from the presence of multiple persistent organic pollutants (POPs), such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), which reduce the activity of activated sludge and are difficult to eliminate. Characterizing and applying active bacterial degraders metabolizing multiple POPs therefore helps to reveal the mechanisms of synergistic metabolism and to improve wastewater treatment efficiency in industrial parks. To date, SIP studies have successfully investigated the biodegradation of PAHs or PCBs in real-world habitats. DNA-SIP facilitates the isolation of target microorganisms that pose environmental concerns. Here, an indigenous phenanthrene (PHE)- and biphenyl (BP)-degrading strain in wastewater, Ralstonia pickettii M1, was isolated via a cultivation-dependent method, and its role in PHE and BP degradation was confirmed by DNA-SIP. Our study provides a routine protocol for the application of DNA-SIP in the isolation of the active functional degraders from an environment.