The endolithic environment is a ubiquitous habitat for microorganisms and a critical interface between biology and geology. In this study, a culture-based method and the phylogenetic analysis based on 16S rRNA and internal transcribed spacer (ITS) sequences were used to investigate the diversity of endolithic bacteria and fungi in two main types of carbonate rocks (namely dolomite and limestone) from Nanjiang Canyon in Guizhou karst area, China. The results of bacterial diversity indicated that all bacteria isolated from dolomite and limestone rocks were divided into 4 bacterial groups, including Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. For these two kinds of rocks, Proteobacteria was the first dominant group, and Gammaproteobacteria occupied the greatest proportion which might be closely related to Pseudomonas in phylogeny to be the most dominant genera after isolation. Actinobacteria and Bacillus bacteria were also widespread in these two kinds of rock environments. There were only 9 and 8 strains of fungi isolated from dolomite and limestone respectively, which all belonged to Ascomycota. To the best of our knowledge, this is the first report on diversity of endolithic culturable bacteria and fungi in carbonate rocks in Guizhou karst region. These microorganisms may play an important and unprecedented role in the carbonate rock weathering during the long history of geological evolution.