A hydrogen-oxidizing bacterium Ideonella sp. TH17 was investigated for ammonium removal from wastewater. Possible pathways and mechanisms of nitrogen assimilation under the stress of two typical quinolone antibiotics and their mixture were studied by molecular biological approaches. The results showed that the capability of ammonium assimilation by TH17 was stimulated at lower concentration (5 μg/L), but inhibited at elevated concentration of quinolone antibiotics. The inhibitory effect of mixed antibiotics was weaker than that of individual antibiotics at higher concentration. Under stress of 5 μg/L quinolone antibiotics, the expression of ammonium assimilation genes was up-regulated, and activities of functional and antioxidant enzymes were enhanced. Meanwhile, the expression of antibiotic resistance gene was up-regulated, which increased the tolerance of TH17 to quinolone antibiotics. Further, amino acid, purine and biotin metabolisms were up-regulated significantly to provide energy, substrates, and coenzymes for enhanced nitrogen metabolism of TH17. Therefore, the influencing pathways and mechanisms of low-concentration quinolone antibiotics on nitrogen removal by TH17 were revealed by gene, enzyme and metabolite molecules, which could guide the development and application of novel processes for biological nitrogen removal from wastewater.