Abstract:[Background] The microorganisms in the Taklimakan Desert as a typical extreme habitat with high temperatures and drought demonstrate distinct regional characteristics after undergoing long-term adaptation to the environment. [Objective] To obtain culturable microbial resources in the extreme environment of the Taklimakan Desert and to explore the effects of different diluted media supplemented with norfloxacin on the isolation of microorganisms in this area. [Methods] Different dilutions of Gauze’s medium No. 1, LB medium, and R2A medium were supplemented with norfloxacin at a final concentration of 0.015 µg/mL for the isolation of microorganisms from the Taklimakan Desert. [Results] A total of 246 strains were isolated and identified, which belonged to 221 species, 58 genera, 29 families, 21 orders, 6 classes of 4 phyla. Firmicutes and Actinobacteria were the dominant phyla, Bacilli and Actinomycetia the dominant classes, Bacillales and Streptomycetales the dominant orders, Bacillaceae and Streptomycetaceae the dominant families, and Streptomyces, Bacillus, and Nocardiopsis the dominant genera. Eighty strains with the maximum similarity lower than 98.65% were classified as potential new species, and 2 strains with the maximum similarity lower than 95.00% were classified as potential new genera. A total of 221 isolated strains were tested for drug sensitivity, among which 43 strains represented by Streptomyces showed resistance to norfloxacin. Twenty-five isolates showed moderate resistance to norfloxacin. [Conclusion] Different dilutions of media supplemented with norfloxacin are beneficial for the isolation of microorganisms in the extreme environment of the Taklimakan Desert. With this method, rich microbial resources were isolated, including a number of new species and drug-resistant strains. The findings provide a basis for the subsequent mining of drug-resistant strains and exploring microbial resources in extreme environments.