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The members of <i>Microbacterium</i> isolated from different environments are known to form peptidoglycan. In this study, we compared the biofilm-forming abilities of <i>Microbacterium</i> sp. PAMC22086 (PAMC22086), which was isolated from the soil in the South Shetland Islands and <i>Microbacterium</i> sp. PAMC21962 (PAMC21962), which was isolated from algae in the South Shetland Islands. The analysis of average nucleotide identity and phylogeny of PAMC22086 revealed a 97% similarity to <i>Microbacterium oxydans</i> VIU2A, while PAMC21962 showed a 99.1% similarity to <i>Microbacterium hominis</i> SGAir0570. For the comparative genomic analysis of PAMC22086 and PAMC21962, the genes related to biofilm formation were identified using EggNOG and KEGG pathway databases. The genes possessed by both PAMC22086 and PAMC21962 are <i>cpdA</i>, <i>phnB</i>, <i>rhlC</i>, and <i>glgC</i>, which regulate virulence, biofilm formation, and multicellular structure. Among the genes indirectly involved in biofilm formation, unlike PAMC21962, PAMC22086 possessed <i>csrA</i>, <i>glgC</i>, and <i>glgB</i>, which are responsible for attachment and glycogen biosynthesis. Additionally, in PAMC22086, additional functional genes <i>rsmA,</i> which is involved in mobility and polysaccharide production, and <i>dksA</i>, GTPase, and <i>oxyR,</i> which play roles in cell cycle and stress response, were identified. In addition, the biofilm-forming ability of the two isolates was examined in vivo using the standard crystal violet staining technique, and morphological differences in the biofilm were investigated. It is evident from the different distribution of biofilm-associated genes between the two strains that the bacteria can survive in different niches by employing distinct strategies. Both strains exhibit distinct morphologies. PAMC22086 forms a biofilm that attaches to the side, while PAMC21962 indicates growth starting from the center. The biofilm formation-related genes in <i>Microbacterium</i> are not well understood. However, it has been observed that <i>Microbacterium</i> species form biofilm regardless of the number of genes they possess. Through comparison between different <i>Microbacterium</i> species, it was revealed that specific core genes are involved in cell adhesion, which plays a crucial role in biofilm formation. This study provides a comprehensive profile of the Microbacterium genus’s genomic features and a preliminary understanding of biofilm in this genus, laying the foundation for further research.