The effects of Phomopsis B3 and organic fertilizer used together on soil improvement during continuous cropping of strawberry and on the growth of strawberry plants were investigated using pot experiments conducted at Nanjing Normal University. The experiment involved five treatments, including a control (CK); (A) organic fertilizer and sterilized solid medium; (B) organic fertilizer and Phomopsis B3; (C) organic fertilizer, Trichoderma viride, Aspergillus niger, and Bacillus subtilis; (D) organic fertilizer, Phomopsis B3, T. viride, A. niger, and B. subtilis. Results showed that the average fresh fruit weight resulting from treatments A, B, C, and D was 1.1 times, 1.4 times, 0.9 times, and 1.1 times that of CK, respectively. Compared with CK, yields from treatments B and A increased by 19.7% and 8.2%, respectively, with lower yields following treatments C and D. Strawberry plants exposed to treatment B grew best; plant height and leaf area following treatment B were greater than for the other treatments. The infection incidence and disease index showed that disease resistance in strawberry plants following treatment B was most significant, leading to the determination that Phomopsis B3 can be used as a biological control agent. Additional results showed that during the growing period, the number of fungi and bacteria initially increased with a subsequent decrease, and was highest during flowering. The number of actinomycetes following treatments A, B, C, and D increased by 7.2%, 160.3%, 124.5%, and 82.6%, respectively, compared with CK during the maturation stage. During flowering, B and D treatments produced the highest percentage increases in soil invertase activity, with treatments A, B, C, and D resulting in increases by 11.1%, 69.4%, 50.3%, and 77.2%, respectively, compared with CK. Treatment B maintained a higher invertase activity throughout the growing period. The flowering stage was a critical growth period for strawberries because of an increased need for nitrogen. Urease activity resulting from treatments A, B, C, and D increased by 250.0%, 700.0%, 250.0%, and 175.0%, respectively, compared with CK. Urease activity following treatment B was greater than for the other treatments during flowering, which helped to improve the conversion of organic nitrogen to effective nitrogen. Soil phosphatase activity following treatments A and C decreased by 67.0% and 46.7%, respectively, while treatments B and D resulted in increases of 122.5% and 227.5%, respectively. Treatment B led to higher soil phosphatase activity throughout the growing period while treatment D resulted in higher phosphatase activity only during flowering. This indicated that treatments B and D, which included Phomopsis B3, can significantly increase soil phosphatase activity. Treatment B produced lower soil cellulase activity during the seedling stage and flowering, but led to higher soil cellulase activity during the fruiting and maturation stages. Phomopsis B3 and organic fertilizer used together can improve soil microflora, improve soil enzyme activity, enhance disease resistance in strawberry plants, and increase strawberry yields. Using Phomopsis B3 and organic fertilizer together might be an effective method to overcome current strawberry cropping obstacles.