[3] The species is known only from the Rhynie chert in Aberdeenshire, Scotland, where it grew in the vicinity of a silica-rich hot spring, together with a number of associated vascular plants such as a smaller species Rhynia gwynne-vaughanii which may be interpreted as a representative of the ancestors of modern vascular plants and Asteroxylon mackei, which was an ancestor of modern clubmosses (Lycopsida).

The spores may therefore be interpreted as meiospores, the product of meiotic divisions, and thus the plants described by Edwards and by Kidston and Lang were diploid, sporophytes.

The plant was originally interpreted as a tracheophyte, because the stem has a simple central vascular cylinder or protostele,[3] but more recent interpretations in the light of additional data indicated that Rhynia major had water-conducting tissue lacking the secondary thickening bars seen in the xylem of Rhynia gwynne-vaughanii, more like the water-conducting system (hydrome) of moss sporophytes.

Aglaophyton is among the first plants known to have had a mycorrhizal relationship with fungi,[5] which formed arbuscules in a well-defined zone in the cortex of its stems.

Aglaophyton lacked roots, and like other rootless land plants of the Silurian and early Devonian may have relied on mycorrhizal fungi for acquisition of water and nutrients from the soil.