The Lascaux Cave in France is an old karstic channel where the running waters are collected in a pool and pumped to the exterior. It is well-known that water bodies in the vicinity of humans are suspected to be reservoirs of amoebae and associated bacteria. In fact, the free-living amoebae Acanthamoeba astronyxis, Acanthamoeba castellanii, Acanthamoeba sp. and Hartmannella vermiformis were identif ied in the sediments of the cave using phylogenetic analyses and morphological traits. Lascaux Cave sediments and rock walls are wet due to a relative humidity near saturation and water condensation, and this environment and the presence of abundant bacterial communities constitute an ideal habitat for amoebae. The data suggest the need to carry out a detailed survey on all the cave compartments in order to determine the relationship between amoebae and pathogenic bacteria.