A series of investigations were conducted using sequencing batch biofilm reactor (SBBR) to explore the influence of C:N:P ratio on biological dephosphatation including the denitrifying dephosphatation and the denitrification process. Biomass in the reactor occurred mainly in the form of a biofilm attached to completely submerged disks. Acetic acid was used as the source of organic carbon. C:N:P ratios have had a significant effect on the profiles of phosphate release and phosphate uptake and nitrogen removal. The highest rates of phosphate release and phosphate uptake were recorded at the C:N:P ratio of 140:70:7. The C:N ratio of 2.5:1 ensured complete denitrification. The highest rate of denitrification was achieved at the C:N:P ratio of 140:35:7. The increase of nitrogen load caused an increase in phosphates removal until a ratio C:N:P of 140:140:7. Bacteria of the biofilm exposed to alternate conditions of mixing and aeration exhibited enhanced intracellular accumulation of polyphosphates. Also, the structure of the biofilm encouraged anaerobic–aerobic as well as anoxic–anaerobic and absolutely anaerobic conditions in a SBBR. These heterogeneous conditions in the presence of nitrates may be a significant factor determining the promotion of denitrifying polyphosphate accumulating organism (DNPAO) development.