Abstract: Microbial carbon fixation and nitrogen fixation play crucial roles in biogeochemical cycles and energy flow in the biosphere. The dark and nutrient-limited environments of caves harbor rich and unique microbial resources. To understand the composition and diversity of carbon-fixing and nitrogen-fixing bacterial communities in karst caves, this study used nine isolation media to selectively culture bacteria from karst cave sediments. Based on 16S rDNA sequencing, the phylogeny and diversity of the isolated strains were analyzed. A total of 230 strains of carbon-fixing bacteria were isolated, belonging to 3 phyla, 16 genera, and 64 species, with dominant genera being Pseudomonas and Acinetobacter. The Shannon-Wiener diversity index for carbon-fixing bacteria was 3.65, the Margalef richness index was 11.58, and the Pielou evenness index was 0.88. A total of 425 strains of nitrogen-fixing bacteria were isolated, belonging to 3 phyla, 14 genera, and 68 species, with dominant genera being Pseudomonas, Acinetobacter, and Cupriavidus. The Shannon-Wiener diversity index for nitrogen-fixing bacteria was 3.43, the Margalef richness index was 11.07, and the Pielou evenness index was 0.81. Additionally, strains isolated on carbon-free medium (CFMD) and nitrogen-free medium (NFb) exhibited the highest diversity. The results indicate the presence of abundant carbon-fixing and nitrogen-fixing bacterial groups in the cave sediments of Libo Karst, providing microbial resources for further research on the functions of these microorganisms in karst cave ecosystems and their potential agricultural applications.