The distribution and bioaccumulation of environmental pollutants are essential to understanding their toxicological mechanism. However, achieving spatial resolution at the subtissue level is still challenging. Perfluorooctanesulfonate (PFOS) is a persistent environmental pollutant with widespread occurrence. The bioaccumulation behavior of PFOS is complicated by its dual affinity for phospholipids and protein albumin. It is intriguing to visualize the distribution preference of PFOS and investigate the differential microenvironment responses at a subtissue level. Herein, we developed a mass-spectrometry (MS)-based spatial multiomics workflow, integrating matrix-assisted laser desorption/ionization MS imaging, laser microdissection, and liquid chromatography MS analysis. This integrated workflow elucidates the spatial distribution of PFOS in mouse cardiac tissue, highlighting its preferential accumulation in the pericardium over the myocardium. This distribution pattern results in greater toxicity to the pericardium, significantly altering cardiolipin levels and disrupting energy metabolism and lipid transport pathways. Our integrated approach provides novel insights into the bioaccumulation behavior of PFOS and demonstrates significant potential for revealing complex molecular mechanisms underlying the health impacts of environmental pollutants.