Cave environments such as those found in UNESCO World Heritage listed Naracoorte Caves in South Australia are at risk of damage by uncontrolled human visitations (tourism). Damages to cave features such as speleothems, rock art and finger flutings can occur because of alterations in the caves’ microclimatic conditions and indigenous microbial community. These damages may discourage further human visitation to caves with adverse socio-economic impacts. Therefore in this study, we have selected caves in Naracoorte with high numbers of human visitations (> 15000 annually) and different configurations; Stick-Tomato (open), Alexandria (closed) and Strawhaven (control, no tourist access) in order to assess the effects of human visitation on cave microflora. Assessments were carried out by culture dependent and independent analyses of sediments obtained from tourist accessible and inaccessible cave areas. Tourist accessible areas had significantly higher bacterial counts (5.31-5.38 Log cfu g-1 soil) than those from tourist inaccessible areas (4.71-4.93 Log cfu g-1 soil) and control cave (1.52 Log cfu g-1 soil) with fungal viable counts being variable. However bacterial and fungal diversity as assessed by 16S rRNA and ITS based fingerprints was substantially higher in tourist accessible cave areas than in inaccessible areas. While cluster analyses of the microbial community profiles showed differences between the caves, the microflora in tourist accessible and inaccessible areas was only different in the open access Stick-Tomato Cave suggesting that cave configuration can play important roles in microbial distribution in caves. Therefore, this study has shown differences in microbial counts, microbial diversity and cluster patterns in show caves compared to control cave and between tourist accessible and inaccessible areas in some cases. Different factors such human access, cave use and configurations could have been responsible for these differences and further work is required for effective quantification of tourism effects in these caves.