Caves are stable environments with characteristics favoring the development of microorganisms. The allocthonous input of organic matter and microbes into the warm Neotropical caves may favor the development of filamentous fungi, including pathogenic species. Histoplasma capsulatum is a pathogenic species commonly found in caves and associated with bat and bird guano. Many Brazilian caves have been historically visited due to scenic and religious tourism. The objective of this study was to perform a microbiology study for a management plan of a show cave in Brazil, focusing on the presence and distribution of pathogenic and opportunistic fungi in the cave. Statistics analysis was used to verify the influence of touristic activity on air-borne fungi spore load. Fungi were isolated from air and guano in Lapa Nova Cave. Samples were obtained through serial dilution, direct and settle plate techniques. For H. capsulatum, samples were incubated in specific media and conditions. Air-borne fungal spore load was compared prior and during visitation and statistically analyzed. A total of 2,575 isolates from the genera Aspergillus, Calcarisporium, Chaetomium, Cladosporium, Curvularia, Emericella, Eurotium, Fusarium, Geotrichum, Gliocladium, Mucor, Purpureocillium, Paecilomyces, Penicillium, Rhizopus and Trichoderma were identified. Histoplasma capsulatum was not isolated from the cave. Eleven opportunistic species were identified. Significant (p<0.05) variations on fungal richness in the air occurred due to cave visitation. Areas of potential microbiologic risks were indicated and management actions suggested. The results suggest a diverse community inhabiting the cave. Possible opportunistic species should be monitored in show caves and microbiota should always be included in the elaboration of cave management plans. This is the first detailed microbiologic study for a management plan of a show cave in the country. It provides relevant information for future management plans.