Wasson's studies in ethnomycology began during his 1927 honeymoon trip to the Catskill Mountains when his bride, Valentina Pavlovna Guercken, a pediatrician, chanced upon some edible wild mushrooms. Fascinated by the marked difference in cultural attitudes towards the fungus in Russia compared to the United States, the couple began field research which led to the writing of Mushrooms, Russia and History published in 1957. 

In the course of their investigations, they mounted expeditions to Mexico to study the religious use of mushrooms by the native population. They became the first westerners to participate in a Mazatec sacred mushroom ritual. In 1957, they published a Life magazine article (Seeking the Magic Mushroom), bringing knowledge of the existence of psychoactive mushrooms to a wide audience for the first time. 

Through his collaboration with Roger Heim, the mushrooms were subjected to scientific study, and Albert Hoffman, using material grown by Heim from specimens collected by the Wassons, identified the chemical structure of the active compounds, psilocybin and psilocin. Two species of mushroom, Psilocybe wassonii Heim and Psilocybe wassonorum Guzman were named in honor of R. Gordon Wasson. Hofmann and Wasson were also the first westerners to collect specimens of the Mazatec hallucinogen Salvia Divinorum, leading to its description as a new species, and bringing it into cultivation outside of Mexico.

Experiences with the magic mushrooms apparently had a profound effect on Wasson, and fungi remained a persistent theme in his work. His next major contribution was a study of the ancient Vedic intoxicant Soma, which he proposed was based on the psychoactive Fly Agaric (Amanita muscaria) mushroom. This was published in 1967 under the title Soma: Divine Mushroom of Immortality. 

His attention then turned to the Eleusian Mysteries, the initiation ceremony of the ancient Greek cult of Demeter and Persephone. In The Road to Eleusis: Unveiling the Secret of the Mysteries (1978), co-authored with Albert Hoffman and Carl A.P. Ruck, it was proposed that the special potion "kykeon", a pivotal component of the ceremony, contained psychoactive ergoline alkaloids from the fungus Ergot (Claviceps spp.).

Prior to his work on Soma, theologians had interpreted the Vedic and Magian practices to have been based on alcoholic beverages that produced inebriation. Wasson was the first researcher to propose that the form of Vedic intoxication was entheogenic.