Once the body dies, the inherent microbes of the host begin to break down from the inside and play a key role thereafter. It is hypothesized that after the death certain rectal microbes would change during the decomposition course in the body. This study aimed to investigate the probable shift in the composition of the rectal flora at different time intervals up to 15 days after death and to explore bacterial taxa important for estimating the time of death. At the phylum level, Proteobacteria and Firmicutes showed major shifts, when checked at 11 different intervals, and emerged at most of the postmortem intervals. At the species level, Enterococcus faecalis and Proteus mirabilis existed at most postmortem intervals; the former showed a downward trend after day 5 postmortem, while the latter showed an upward trend. There were obvious differences in bacterial community structure and richness at the phylum, genus, and species levels during the decomposition of the corpse of rats. The phylum, genus, and species taxa richness decreased initially and then increased significantly. The turning point came on day 9 when genus, rather than phylum or species, contained the most information for estimating the time of death. We constructed a prediction model using genus taxon data from high-throughput sequencing, which explained 87.2% of the time since the first sampling within 1 h. Seven bacteria, namely Enterococcus, Proteus, Lactobacillus, unidentified Clostridiales, Vagococcus, unidentified Corynebacteriaceae, and unidentified Enterobacteriaceae, were included in this model. The above-mentioned bacteria showed a promising future for estimating the shortest time of death and results of current study were agreeing with the proposed hypothesis.