Ammonia-rich lagoon supernatant was treated using anammox process in an integrated fixed-film activated sludge (IFAS) laboratory reactor. Effective anammox activities were demonstrated over 259 days of operation. The ammonium removal efficiency reached 94% in Phase I with influent concentrations of NH4+, NO2- and chemical oxygen demand (COD) at 250 mg-N/L, 325 mg-N/L, and 145 mg-COD/L, and reached 88% in Phase II at 420 mg-N/L, 525 mg-N/L, and 305 mg-COD/L. When supplemented with nitritation effluent for nitrite sources in Phase III, the influent COD concentration increased to 583 mg-COD/L without loss of ammonia removal efficiency (87%). The specific anammox activity was higher in biofilm than in the suspended flocs (P < 0.05), increased from Phase I to II (P < 0.05), and decreased in Phase III. Ammonia removal related genes were quantified using qPCR. Results showed higher anammox gene (AMX nirS) prevalence in biofilm, while denitrification genes (nosZ and narG) were higher in flocs (P < 0.05). Microbial community analysis showed that the seeded anammox bacteria Candidatus Brocadia was maintained at 19% in the biofilm and only 0.3% in the flocs. The major taxa in the flocs were related to denitrifiers. The floc community was affected largely under high COD conditions, but the biofilm community was not. These results suggest that the anammox activity in biofilm is resilient to high COD loadings, due to the existence of flocs with denitrification activity. The segregation of bacterial communities between biofilm and flocs in the anammox IFAS system resulted in high ammonia removal efficiency and resistance to high organic loadings.