The microbiota is the community of microorganisms living on and in a biological system. It has shown to play a role in a broad range of medical conditions, including cardiovascular diseases. Germ-free (GF) mice grew under the condition lacking all microorganisms, in contrast to conventionally raised (CONV-R) mice colonized with a diverse microbiota. GF mice play a key role as tools to reveal the the causal relationship between microbiome and disease. In particular, GF mice present significant cardiac functional defects compared to CONV-R mice. The project's goal was to determine the spatial impact of GF vs. CONV-R status on cardiac metabolism across cardiac regions. 4 metabolite molecules showed significant differences by Random Forest analysis based on untargeted liquid chromatography-tandem mass spectrometry data. The four molecules are neighborhood of ADP-ribose (m/z = 588.11), ergothioneine (m/z = 230.1), neighborhood of dodecanoic acid (m/z = 177.13), and L-glutamine (m/z = 130.05). (Neighborhood results were generated from molecular networking.) Parallel reaction monitoring (PRM) was applied to provide more precise quantification results. In most locations, peak abundance significant difference was mapping in the locations: right ventricle free wall and left atrium part between germ-free and CONV-R conditions. Ergothioneine showed a difference in the left ventricle free wall bottom section. The metabolites may relate to the presence of microbiome and contribute to reducing the risk of cardiovascular disease.