This study concerns total arsenic (TAs) and arsenic species determination in three species of mushrooms collected in Yunnan, China. The purpose of this study was to check concentration level of arsenic in Boletus edulis, Tricholoma matsutake and Suillellus luridus, estimate arsenic bioaccessibility and find out which arsenic species occur in mushrooms to assess if they may pose a threat to human health. An analytical methodology based on ion chromatography (IC) hyphenated to inductively coupled plasma mass spectrometry (ICP-MS) with dynamic reaction cell (DRC) and size exclusion chromatography (SEC) with UV-Vis detection and ICP-DRC-MS detection. Ultrasound assisted extraction (UAE), microwave assisted extraction (MAE) and enzymatic assisted extraction (EAE) were applied. Quantification of As species in extracts was performed by IC/ICP-DRC-MS in the first dimension. Slightly better extraction efficiencies were obtained for MAE (from 75% to 90%) then for UAE. EAE was used for estimation of bioaccessibility by application of a modified BARGE bioaccessibility method (UBM) for in vitro studies. Bioaccessibility values were in the ranges of 73%-102%, 74%-115% and 18%-87% for step 1 (S1), for step 2 (S2) and for step 3 (S3) of EAE, respectively. Extracts obtained after EAE were subjected to SEC-UV-Vis/ICP-DRC-MS analysis as the second dimension. The main signal was obtained in the area of a molecular mass of ∼5 kDa for all mushroom extracts. Monitoring of an 50SO+ ion confirmed that this signal comes from As-protein. In sample of Boletus edulis additional signal occurred which is classified as unknown As-compound. Both signals require identification with another analytical technique.