Limestone areas across the world develop karstic caves, which are populated by a wide range of macro‐ and microorganisms. Many of these caves display Paleolithic art or outstanding speleothems, and in the last century they have been subjected to anthropization due to touristic management and intense human frequentation. Despite their cultural importance and associated conservation issues, the impact of anthropization on cave biodiversity is not known. Here, we show that anthropization is associated with specific cave biota modifications. We compared diversity in four pristine caves, four anthropized show caves, and the iconic Lascaux Cave with even stronger anthropization. The predominant microbial higher taxa were the same in all caves, but the most anthropized cave (Lascaux) was unique as it differed from the eight others by a higher proportion of Bacteroidetes bacteria and the absence of Euryarchaeota and Woesearchaeota archaea. Anthropization resulted in lower diver‐sity and altered community structure for bacteria and archaea on cave walls, espe‐cially in Lascaux, but with a more limited effect on microeukaryotes and arthropods. Our findings fill a key gap in our understanding of the response of karstic communi‐ties to anthropization, by revealing that tourism‐related anthropization impacts on the prokaryotic microbiome rather than on eukaryotic residents, and that it shapes cave biota irrespective of cave natural features.