Elevated levels of potentially toxic elements (PTEs) have been frequently detected in substrates used for cultivating edible mushrooms. However, the distribution and accumulation pathways of PTEs within mushrooms and their cultivation substrates remain inadequately understood. This study specifically aims to clarify these aspects of button mushrooms (Agaricus bisporus). A systematic analysis was conducted on the distribution of PTEs (As, Cd, Cr, Cu, Ni, Pb, and Zn) and stable Pb isotope fingerprints across various components of the button mushroom cultivation systems, including the cultivation substrates (a mixture of wheat straw, chicken manure, soil, and additives), fruiting bodies (MR), and spent mushroom substrate (SMS). The methodology involved inductively coupled plasma mass‒spectrometry for PTE quantification and Pb isotope ratio analysis for source identification. The results demonstrated that wheat straw and additives were the main contributors to the endogenous accumulation of PTEs in MR, accounting for 35.0% and 33.2% of Pb accumulation, respectively. Furthermore, MR, wheat straw, and soil together contributed to more than 67% of the Pb loads in SMS. The study also revealed that the occurrence of PTEs in these substrates is predominantly driven by exogenous sources, particularly the significant contribution from anthropogenic activities. Overall, these findings provide valuable biogeochemical insights into the accumulation pathways and patterns of PTEs in button mushroom cultivation systems, with potential applications for cleaner food management and environmentally sustainable practices.