Marimo (lake ball) is an uncommon ball-like aggregation of the green alga, Aegagropila linnaei1. Although A. linnaei is broadly distributed in fresh and brackish waters in the northern hemisphere, marimo colonies are found only in particular habitats. The colonies have been gradually shrinking in recent years. Nevertheless, it is not clear how and why A. linnaei forms such massive spherical aggregations. Here, we report the bacterial microbiomes inside various sizes and aggregating structures of natural marimo collected from Lake Akan, Japan. We observed multi-layers composed of sediment particles only in the sizeable radial-type marimo with a >20 cm diameter, not in the tangled-type marimo. The deeper layers were enriched by Nitrospira, potential novel sulphur-oxidizing bacteria, and sulphate-reducing Desulfobacteraceae bacteria. The sulphur cycle-related bacteria are unique to Lake Akan due to sulphur deposits from the nearby volcanic mountains. Some of them were also recovered from lake sediments. Microorganisms of the multi-layers would form biofilms incorporating nearby sediment, which would function as microbial seals within large radial-type marimo. We propose that the layer structure provides habitats for diverse bacterial communities, promotes airtightness of the marimo, and finally contributes to the massive growth of the aggregation. These findings provide a clue to deciphering the massive growth of endangered marimo aggregates.