MotivationAccurate determination and quantification of the taxonomic composition of microbial communities, especially at the species level, is one of the major issues in metagenomics. This is primarily due to the limitations of commonly used 16S rRNA reference databases, which either contain a lot of redundancy or a high percentage of sequences with missing taxonomic information. This may lead to erroneous identifications and, thus, to inaccurate conclusions regarding the ecological role and importance of those microorganisms in the ecosystem.ResultsThe current study presents MIMt, a new 16S rRNA database for archaea and bacteria’s identification, encompassing 47 001 sequences, all precisely identified at species level. In addition, a MIMt2.0 version was created with only curated sequences from RefSeq Targeted loci with 32 086 sequences. MIMt aims to be updated twice a year to include all newly sequenced species. We evaluated MIMt against Greengenes, RDP, GTDB and SILVA in terms of sequence distribution and taxonomic assignments accuracy. Our results showed that MIMt contains less redundancy, and despite being 20 to 500 times smaller than existing databases, outperforms them in completeness and taxonomic accuracy, enabling more precise assignments at lower taxonomic ranks and thus, significantly improving species-level identification.