A series of atmospheric investigations was conducted in Carlsbad Cavern to determine if human visitation is a possible cause for the contamination of the cave system with non-indigenous microorganisms. In 2004, site-specific culture-based data demonstrated that Staphylococcus spp. colony-forming units (CFUs) were the most prevalent members of the atmospheric community along the paved visitor trail (avg. 18.8% of CFU), while Knoellia spp. CFUs dominated off-trail locations (40.1% of CFU). Fungal culture data revealed that Penicillium and Aspergillus were prevalent in the Lunch Room where food is stored, sold, and consumed. Ubiquitous genera such as Cladosporium and Alternaria were prevalent near the Natural Entrance of the cave, and the general trend was a decrease in fungal CFUs with progression into the cave system, except for the area near the Lunch Room. Management practices such as prohibition of crumb-generating types of foods could be considered to protect cave health. In 2009, nonculture-based analyses demonstrated that Enterobacteriaceae were the dominant microbiota at sites along the descent trail and within the Lunch Room. Dominance of Enterobacteriaceae has not been previously demonstrated in caves. Either they are naturally occurring indigenous members, or their presence is a marker of anthropogenic contamination.