Bacterial diversity in sediments at UNESCO World Heritage listed Naracoorte Caves was surveyed as part of an investigation carried out in a larger study on assessing microbial communities in caves. Cave selection was based on tourist accessibility; Stick Tomato and Alexandra Cave (> 15000 annual visits) and Strawhaven Cave was used as control (no tourist access). Microbial analysis showed that Bacillus was the most commonly detected microbial genus by culture dependent and independent survey of tourist accessible and inaccessible areas of show (tourist accessible) and control caves. Other detected sediment bacterial groups were assigned to the Firmicutes, Actinobacteria and Proteobacteria. The survey also showed differences in bacterial diversity in caves with human access compared to the control cave with the control cave having unique microbial sequences (Acinetobacter, Agromyces, Micrococcus and Streptomyces). The show caves had higher bacterial counts, different 16S rDNA based DGGE cluster patterns and principal component groupings compared to Strawhaven. Different factors such as human access, cave use and configurations could have been responsible for the differences observed in the bacterial community cluster patterns (tourist accessible and inaccessible areas) of these caves. Cave sediments can therefore act as reservoirs of microorganisms. This might have some implications on cave conservation activities especially if these sediments harbor rock art degrading microorganisms in caves with rock art.