The Naica Underground System (NUS) in Northern Mexico comprises a lead, zinc, and silver producing mine and displays the largest gypsum crystals ever found in natural caves. The caves are now closed to the public and mining activities have been suspended for an undefined period since October 2015. Besides its geological, economical, and tourist importance, the bacterial diversity in the NUS has not been fully explored yet. This study surveyed for bacteria present on different mineral substrates (gypsum crystals, iron oxide crusts) and hot spring samples collected before the NUS was inaccessible, using culture-dependent and culture–independent (PCR-DGGE) methods. This study is the first reporting the isolation of microorganisms from Naica. Cluster analysis of DGGE fingerprints revealed slight differences between communities from caves and tunnels and according to their mineral substrate type while communities from solid substrates and water samples appeared to be more distant. Both approaches, culture-dependent and independent, revealed the presence of bacteria from the Firmicutes, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria in gypsum crystals, iron oxide crusts, and hot springs, respectively. Deinococcus-Thermus and Actinobacteria were only detected by DGGE in hot spring samples. According to 16S rRNA sequencing, heterotrophic bacteria isolated under aerobic conditions were affiliated with Bacillus, Brevibacillus, Paenibacillus, Schlegelella, Cupriavidus, Pseudoxanthomonas, and Lysobacter. Most of the isolates and sequences retrieved by DGGE were related to organisms previously detected in other extreme subsurface environments. Some of the isolates were able to precipitate calcium carbonate and precipitate Fe(III) in solid media but their possible participation in biomineralization processes in situ has still to be investigated. Microbial communities found in the NUS are likely autochthonous with some allochtonous components due to human intervention. Their role in geobiological processes requires further investigation.