Given the limited capacity of natural forests for wood production and their long rotation period, fast-growing species are considered suitable alternatives to meet timber demands. Studying the effects of afforestation with different species on soil biological indicators, understory regeneration, and the physical and chemical properties of soil is essential for understanding ecological succession, ecosystem restoration, and biodiversity conservation. In this study, the effects of Paulownia cultivation that recognized as one of the most debated species for wood supply in Iran on soil biological properties were investigated. For this purpose, a Paulownia plantation located in Malayer County was selected, and a complete inventory of all trees was conducted. To evaluate soil biological indicators, ten soil samples were collected from the 0–15 cm depth. The mean diameter and height of the trees were 15.41 cm and 3.38 m, respectively. Prior to statistical analyses, data normality was assessed using the Kolmogorov–Smirnov test. Independent t-tests were then applied to compare soil biological indicators between the Paulownia plantation and the control site. Results revealed that basal respiration (mean: 2.31 in Paulownia stand and 1.73 in control) and substrate-induced respiration (25.8 and 23.6, respectively) showed no significant difference, whereas nitrification potential (217.7 and 80.7) and microbial biomass carbon (6.37 and 3.17) exhibited significant differences at the 1% level. Overall, microbial biomass carbon and nitrification potential were higher in the Paulownia plantation compared to the control site. These findings, reported for the first time in this context, indicate that Paulownia cultivation can improve certain soil biological parameters. However, to achieve a more comprehensive understanding and to strengthen and generalize the obtained results to large-scale afforestation, further complementary studies are recommended.