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Aims: This study assessed the use of matrix‐assisted laser desorption/ionization time of flight (MALDI‐TOF) mass spectrometry as an alternative method to identify species associated with the thanatomicrobiota and epinecrotic communities. Methods and Results: The study was conducted on 10 murine cadavers, and microbiological swabs were collected from five external anatomical sites (eyes, ears, nose, mouth and rectum) and four internal organs (brain, spleen, liver, heart), during 16 and 30 days, for the thanatomicrobiota and epinecrotic communities, respectively. Our results revealed that the postmortem microbiota associated with the external cavities showed changes over time and reduced taxonomic diversity. The internal organs, initially sterile, showed signs of microbial invasion at 3 and 10 days postmortem for the liver‐spleen and heart‐brain, respectively. The postmortem microbiota was mainly dominated by Firmicutes and Proteobacteria. Conclusions: MALDI‐TOF is a promising method for estimating postmortem interval (PMI), associated with rapid sample handling, good reproducibility and high productivity. Significance and Impact of the Study: This study investigated microbial changes during the decomposition process and proposed a simple strategy for PMI estimation. Results introducing the application of the MALDI‐TOF method in the field of forensic. [ABSTRACT FROM AUTHOR]