The radioactive isotope, ⁴⁰K, of naturally occurring potassium (0.012%) is present in the Earth’s crust in a low percentage of all potassium, leading to its presence in almost all foodstuffs. The impact of ⁴⁰K activity concentrations was assessed in wild and cultivated edible mushrooms and in growing substrates. Samples were analysed by gamma spectroscopy. In the wild mushroom species, the average activity concentration of ⁴⁰K was 1291 Bq kg⁻¹ dry weight (dw), approximately 140 Bq kg⁻¹ fresh weight (fw), with a range of average values per species from 748 in Lactarius deliciosus to 1848 Bq kg⁻¹ dw in Tricholoma portentosum. The cultivated species presented an average value of 1086 Bq kg⁻¹ dw; and the soils, compost of cultivation and wood of substrate are 876, 510 and 59.4 Bq kg⁻¹ dw, respectively. The total K content reached a maximum of 59,935 mg kg⁻¹ dw in T. portentosum. The transfer factors (TF > 1) suggested that mushrooms preferentially bioconcentrated ⁴⁰K. Cantharellus cibarius, Craterellus tubaeformis, Hydnum repandum and T. portentosum by most TF could be considered as bioindicators of ⁴⁰K. Taking into account that the annual radiation dose of ⁴⁰K due to the average consumption of mushrooms analysed (0.15 μSv/year) is very low, it can be concluded that the consumption of these mushrooms does not represent a toxicological risk for human health. Finally, according to the total K content, from the nutritional point of view, these mushrooms could be considered as a potential source of potassium for the human diet.