Naked barley is one of the major crops of western China. Root rot causes a serious reduction in naked barley. However, study of the rhizosphere soil microorganisms and enzyme activity of naked barley experiencing root rot may yet find ways to mitigate the damage. This study selected a naked barley growing area in Zhuoni county of the Gannan state in Gansu Province as its research site. Naked barley root rot was chosen as the object of study. Rhizosphere soil samples from healthy naked barley and naked barley infected with root rot were collected separately. Then microbial (bacteria, actinomyces, fungi) quantity was determined using the plate count method. Microbial biomass carbon was determined using the potassium dichromate sulfuric acid heating method. Microbial biomass nitrogen was determined using the Kjeldahl method. Microbial biomass phosphorus was determined using the molybdenum blue colorimetric method. Catalase activity was determined using the volumetry method. Sucrase and cellulase activity were determined using 3, 5-dinitrosalicylic acid colorimetry. Urease activity was determined using the indophenol blue colorimetric method. Alkaline phosphatase was determined using the phenyl phosphate disodium colorimetric method. Finally, we analyzed rhizosphere soil sampled from healthy naked barley and from naked barley infected with root rot to compare microbial biomass, microbial numbers in soil, and soil enzyme activity. The results showed that root rot occurred in naked barley at all 10 of the sampled areas with an incidence of 5% to 20%, though the morbidity differed between plots. The presence of root rot dramatically affected the microbial biomass of naked barley rhizosphere soil, which led to changes in the carbon, nitrogen, and phosphorus levels of the microbial biomass. The reduction in microbial biomass nitrogen and phosphorus levels and in microbial biomass differed between sampling sites. The total number of microorganisms varied, but showed a general population trend of bacteria > actinomyces > fungi. Different microorganisms have different responses to naked barley root rot, as the number of bacteria and actinomyces decreased when root rot was present, while the number of fungi increased. The total number of microorganisms varied between different sample areas, with bacteria and fungi showing rationality, while actinomyces did not. The presence of naked barley root rot also changed the enzyme activity of rhizosphere soil. Enzyme activity around sucrose, urease, and alkaline phosphatase decreased in the presence of root rot, while enzyme activity around cellulose increased. Changes in catalase activity did not appear to be correlated with the presence of root rot. Enzyme activity differed between samples of naked barley rhizosphere soil different samples to a significant degree. Correlation analysis showed a significant positive correlation between soil microbial biomass carbon and soil microbial biomass nitrogen levels. The number of soil bacteria and actinomyces showed a significant positive correlation with both soil microbial biomass carbon and biomass nitrogen levels. Soil fungi numbers showed significant positive correlation with both soil microbial biomass phosphorus and sucrose levels, and with enzyme activity around cellulose. Alkaline phosphatase activity showed significant positive correlation with both soil microbial biomass carbon and actinomyces numbers, but also showed significant negative correlation with catalase activity. In general, the presence of naked barley root rot was found to influence various factors of rhizosphere soil. These various factors are connected to and influence each other. The root rot changed the soil microbial flora composition of naked barley rhizosphere soil by restricting the presence of materials such as carbon, nitrogen, and phosphorus, and by disrupting energy metabolism. Therefore, research into the control of naked barley root rot must take the role of soil microorganisms and enzymes into account.