Microbes　represent　the　most　common　organisms　on　Earth;　however,　less　than　2%　of　microbial　species　in　the　environment　can　undergo　cultivation　for　study　under　laboratory　conditions,　and　the　rest　of　the　enigmatic,　microbial　world　remains　mysterious,　constituting　a　kind　of　＂microbial　dark　matter＂　(MDM).　In　the　last　two　decades,　remarkable　progress　has　been　made　in　culture-dependent　and　culture-independent　techniques.　More　recently,　studies　of　MDM　have　relied　on　culture-independent　techniques　to　recover　genetic　material　through　either　unicellular　genomics　or　shotgun　metagenomics　to　construct　single-amplified　genomes　(SAGs)　and　metagenome-assembled　genomes　(MAGs),　respectively,　which　provide　information　about　evolution　and　metabolism.　Despite　the　remarkable　progress　made　in　the　past　decades,　the　functional　diversity　of　MDM　still　remains　uncharacterized.　This　review　comprehensively　summarizes　the　recently　developed　culture-dependent　and　culture-independent　techniques　for　characterizing　MDM,　discussing　major　challenges,　opportunities,　and　potential　applications.　These　activities　contribute　to　expanding　our　knowledge　of　the　microbial　world　and　have　implications　for　various　fields　including　Biotechnology,　Bioprospecting,　Functional　genomics,　Medicine,　Evolutionary　and　Planetary　biology.　Overall,　this　review　aims　to　peel　off　the　layers　from　MDM,　shed　light　on　recent　advancements,　identify　future　challenges,　and　illuminate　the　exciting　opportunities　that　lie　ahead　in　unraveling　the　secrets　of　this　intriguing　microbial　realm.