We present the case of a 53-year-old woman who was employed at a mushroom (Pleurotus eryngii and Hypsizigus marumoreus) cultivation factory for 15 years. She was admitted to our hospital because of fever and dry cough. Chest radiography and CT scanning revealed diffuse ground glass opacity and centrilobular nodules in both lung fields. Serum KL-6 was elevated. In the bronchoalveolar lavage fluid, the CD4/CD8 ratio was reduced, and the lymphocyte fraction was very high. Transbronchial lung biopsy specimens showed lymphocyte alveolitis. After admission, the patient's symptoms improved rapidly without medication. Although these findings are compatible with hypersensitivity pneumonitis, it was difficult to identify a causative antigen. Serum antibody against Trichosporon was positive. A lymphocyte stimulation test of the peripheral blood was positive against extracts of P. eryngii and H. marumoreus. Furthermore, precipitins against the extracts of H. marumoreus were detected by a double immunodiffusion test. Therefore, we decided to conduct a challenge test using H. marumoreus. As an inhalation provocation test with H. marumoreus conducted in a sickroom caused the same clinical symptoms and signs as experienced in the workplace, we diagnosed hypersensitivity pneumonitis caused by H. marumoreus. A provocation test, in which antigen exposure is limited using a closed space, such as a sickroom, was simple, safe and effective for determining the antigen causing hypersensitivity pneumonitis.