The study investigated species composition and polysaccharides, proteins, and eDNA content in EPS fractions (soluble, Sol-EPS; loosely bound, LB-EPS; tightly bound, TB-EPS) in nitrifying aerobic granules from reactor operated at a high load of nitrogen 0.5 kg TKN/(m3 × day). In the study, polysaccharides predominated in Sol-EPS, whereas proteins were the main component of bound EPS. eDNA was only detectable in TB-EPS. In Sol-EPS, eDNA originating from Pseudomonales predominated; species belonging to Pseudomonales produce glue-like polysaccharides that enable surface colonization. In all EPS fractions, high abundance of Acinetobacter sp. was noted. In TB-EPS, Thauera sp. was present in high abundance (25.6%) that produce polymers ensuring compact granule structure and that participate in many key metabolic processes for nitrogen conversions in wastewater treatment plants such as heterotrophic nitrification or denitrification. The study indicates that each EPS fraction in aerobic granules represents micro-environment facilitating the growth of species that produce a component of EPS with function essential for surrounding cells.