Display omitted •Anammox bacteria revealed robust resistance to the toxicity to 200 mg/L 1,4-dioxane.•1,4-Dioxane inhibition to anammox process was found to be substrate dependent.•Inactivation of HDH enzyme is the main anammox inhibition mechanism by 1,4-dioxane.•Candidatus Brocadia was adversely affected by increasing 1,4-dioxane concentration. Anammox process contaminated by 1,4-dioxane was evaluated using sets of batch experiments under different nitrogen loads and 1,4-dioxane concentrations. Toxicity of 1,4-dioxane to anammox consortia was negligible till the level of 200 mg-dioxane/L with a reduction in the total nitrogen (TN) removal efficiency, less than 8% at initial loads of <200 mgTN/L. However, 1,4-dioxane inhibition was also found to be substrate-dependent, where TN removal efficiency was declined by 19.3% at 500 mgTN/L and 200 mg-dioxane/L. The negative synergistic effect was exacerbated in the case of 500 mgTN/L and 2000 mg-dioxane/L, with the minimum TN removal efficiency of 24.1 ± 1.2%. Moreover, the impact of 1,4-dioxane on anammox activity was evaluated in terms of specific anammox activity (SAA), hydrazine dehydrogenase (HDH) activity, and extracellular polymeric substances (EPS). These parameters revealed a significant reduction when 1,4-dioxane surpassed 200 mg/L. Their lowest values of 0.90 ± 0.02 mgTN/gVSS/h (54.7% lower than control), 0.45 ± 0.03 µmol cytochrome-c reduced/min/mg protein (45.1% lower than control), and 74.8 ± 8.5 mg/gVSS (32.6% lower than control) were recorded when anammox bacteria were subjected to initial TN and 1,4-dioxane loads of 200 and 2000 mg/L, respectively. The inhibition mechanism of 1,4-dioxane on the anammox process was expected owing to HDH enzyme inactivation that led to a reduction in SAA and EPS secretion ability. Further, the 16S rRNA analysis revealed that the exposure to 1,4-dioxane caused a significant drop in the dominant anammox genus Candidatus Brocadia, along with a progression of denitrifying bacteria, particularly genus Limnobacter.