Utilizing the spatio-temporal intergenerational methodology, we conducted an investigation into the impacts of prolonged fallowing on the composition and functional genetic alterations of the primary microbial communities engaged in the carbon fixation and nitrogen metabolism pathways within the regional soil. To accomplish this, we employed microbial metagenome sequencing technology and utilized the carbon fixation and nitrogen metabolism pathways from the KEGG database as a tool. The study was carried out in sample plots located in the Minqin Oasis, situated at the southeastern edge of the Badan Girin Desert. These plots were categorized into nine groups based on the duration of fallowing:plots with no fallowing, 1-year fallowing, 2-year fallowing, 4-year fallowing, 8-year fallowing, 13-year fallowing, 20-year fallowing, 30-year fallowing, and 40-year fallowing. The findings revealed significant alterations in the abundance of soil microorganisms and functional genes related to carbon fixation and nitrogen metabolism due to fallowing. Bacteria played a predominant role in both carbon fixation and nitrogen metabolism. The primary carbon fixation pathways of soil microorganisms in the study area were the reductive tricarboxylic acid cycle pathway, reductive acetyl-CoA pathway, and the 3-Hydroxypropionate/4-hydroxybutylate cycle pathway. As for nitrogen metabolism, the main pathways identified were anaerobic ammonium oxidation, dissimilatory nitrate reduction, assimilatory nitrate reduction, denitrification, and nitrification. Gemmatimonas, unclassified Chloroflexi, and Streptomyces were main bacteria for carbon fixation of regional soil microorganisms, while the nitrogen metabolism relied mainly on Nitriliruptor, Gemmatimonas, Solirubrobacter, unclassified Chloroflexi, and Streptomyces. Gemmatirosa, unclassified Conexibacter, unclassified Candidatus Rokubacteria, Gaiella, and Geminicoccus could be used as marker microbial populations of the pathways of carbon fixation in the cultivated land in the study area, and coxL.cutL and ACO.acnA were main response function genes of the pathways of carbon fixation in the cultivated land in the study area. Seven genera of classified soil microorganisms, including Nitriliruptor, unclassified Candidatus Rokubacteria, Geminicoccus, unclassified Conexibacter, Solirubrobacter, unclassified Acidobacteria, and Rubrobacter, could be used as the marker microbial population of the pathways of nitrogen metabolism in the study area, and GDH2 and E1.4.7.1 were main response function genes of the pathways of nitrogen metabolism in the study area. These results hold significant importance in elucidating the processes of carbon and nitrogen cycling in the soils of Minqin Oasis under the influence of fallowing.