Using the tobacco variety Yunyan87 as an example, we investigated the effects of the combined application of organic fertilizer and microbial agents on the physicochemical properties and enzyme activities of tobacco soil and on tobacco agronomic parameters. Compared with the control group, the contents of alkali-hydrolyzed nitrogen, available potassium, and available phosphorus in the soil treated with organic fertilizer and microbial agents increased by 27.7, 12.3, and 133.7%, respectively. The activities of soil urease, phosphatase, sucrase, and catalase increased by 86.3, 85.1, 46.6, and 33.5%, respectively, while the numbers of bacteria and actinobacteria increased by 89.9 and 183.4% respectively. In contrast, the numbers of fungi decreased by 64.8%. Plant height, effective leaf number, stem circumference, stalk width, cutting length, and cutting width increased by 8.1, 7.8, 12.1, 15.7, 20.5, and 11.5%, respectively. Based on our results, the application of organic fertilizer combined with microbial agents significantly improved soil properties, enzyme activities, microbial numbers, and tobacco agronomic parameters.