Amanita muscaria contains a number of biologically active agents, at least two of which are known to be psychoactive. Muscimol (3hydroxy-5-aminomethy-1 isoxazole, an unsaturated cyclic hydroxamic acid) is the most significant. It is the product of the decarboxylation or drying of ibotenic acid, another important compound in the biochemistry of the fly agaric. Muscarine, discovered in 1869, was long thought to be the active hallucinogenic agent in A. muscaria until the mid 20th century, when researchers in England, Japan, and Switzerland recognized that these effects were due mainly to ibotenic acid and muscimol.

Fresh fly agaric mushrooms contain choline, acetycholine, muscarine, muscaridine, muscazone, large amounts of ibotenic acid, very little muscimol, and the rare trace elements selenium and vanadium. As a result of the decarboxylation of ibontenic acid, dried fly agarics contain high amounts of muscimol, which is responsible for the psychoactive effects. The pigment is a derivative of the ibotenic acid. The amount of Muscarine is 0.0003% at most. The amount of ibotenic acid in fresh specimens from Germany and Switzerland averaged 0.03% but can run as high as 0.1%. Traces of bufotenine and the tropane alkaloid L-hyoscyamine have also been reported.

Muscimol is regarded as the actual psychoactive constituent, although this view is not without controversy. Nevertheless, muscimol can be detected in the urine of people who have ingested fly agarics. Several experiments have demonstrated that the urine from a person who has consumed fly agric will produce psychoactive effects in other people. Theodore Schurr, an anthropologist and molecular biologist, has summarized our current pharmacological understanding of Siberian fly agaric shamanism:

Once ingested, the psychoactive alkaloids and substances in [Armanita muscaria] acted as agonists of normal brain neurotransmitter function, disrupting the coordinate action between the catecholaminergic and serotoninergic systems and producing hallucinogenic effects similar to those generated by LSD and harmine.

Ibotenic acid and muscimol are structurally related to each other and to two major neurotransmitters of the central nervous system: glutamic acid and GABA respectively. Ibotenic acid and muscimol act like these neurotransmitters (muscimol is a potent GABAA agonist, while ibotenic acid is an agonist of NMDA glutamate receptors and certain metabotropic glutamate receptors) which are involved in the control of neuronal activity. It is these interactions which are thought to cause the psychoactive effects found in intoxication. Following ingestion some of the ibotenic acid is metabolised to muscimol which would appear to be the agent responsible for the majority of the psychoactivity. Indeed, ibotenic acid's strong water solubility means that it could not distribute into the brain without an active process like a transporter; this, and the lack of any reports of permanent brain damage following A. muscaria ingestion (which would be the result of ibotenic acid entering the brain due to its effect as an NMDA receptor agonist) make it unlikely that ibotenic acid enters the brain following A. muscaria ingestion. However, it is worth noting that no studies have directly investigated ibotenic acid's ability to permeate the brain.

Muscazone is another compound more recently isolated from European specimens of the fly agaric. It is a product of the breakdown of ibotenic acid by ultra-violet radiation. It is of minor pharmacological activitycompared with the other agents.

Muscarine binds with Muscarinic acetylcholine receptor and lead to the excitation of the neurons bearing these receptors. The levels in Amanita muscaria are minute when compared with other poisonous fungi, such as the deadly Inocybe e rubescens or small white Clitocybe species C. dealbataand C. rivulosa, and are too insignificant to play a role in the symptoms of poisoning.

Amanita muscaria and related species are known as effective accumulators of vanadium (up to 500 mg/kg in dry weight). Vanadium is present as an organometallic compound (called amavadine) in fruit-bodies. However, the biological importance of the accumulation process is unknown.