Background: The chief aim of this study was to enlist the ethnopharmacological aspect of mushrooms from four Provinces of Pakistan. Due to the seasonal variation and extreme geographical conditions, Pakistan has great mushroom diversity. Mushrooms have been used by the indigenous people for the treatment of different ailments in many regions of Pakistan. They still depend on the mushrooms for treatment of various ailments. Moreover, mushrooms are used as first aid to treat cold, cough, influenza, asthma, cancer, gastric and hepatic disorders. Traditional uses of medicinal mushrooms lead to the discovery of alternative source of synthetic medicines. This is basically the first quantitative ethnopharmcological documentation of medicinal mushrooms from Pakistan. Materials and Method: Ethnopharmacological information was collected from the 950 informants including male and female. The informative data was based on semi-structured interviews, group discussions, and field visits. Data was analyzed by applying different quantitative indices such as informant consent factor (ICF), Use value (UV), Relative Frequency of Citation (RFC), the Fidelity level (FL) and Jaccard Index (JI). Result: Almost 50 mushrooms belonging to 24 families were reported. Most-frequently cited families were Agaricaceae (13 species) and Morchellaceae (5 species). Extract was the common mode of preparation reported (63.33%), followed by Powder (36.67%). Mostly these medicinal mushrooms were present as wild (62.90%), followed by cultivated (37.10%). Among all 11.75% mushrooms were toxic and inedible, 75% were nontoxic, whereas the remaining 13.46% may be toxic or nontoxic because of their dual attitude. New medicinal uses of 23 mushrooms were explored in this study. Conclusion: This ethnopharmacological documentation revealed that the mushrooms are still used by natives of rural area in their day-to-day lives. This study provides the basis for the conservation of mushrooms. Mushrooms with high ICF, UV and FL were further used for phytochemical and pharmacological studies. This documentation is baseline information which can be used to develop new mushroom-based medicines.