Root interactions are crucial in regulating soil microbial metabolism and plant nutrient allocation strategies, especially in mixed plantings. However, the effects of mixed planting and direct root contact on soil properties and plant nutrient allocation remain unclear. Thus, we established potted plants with Moso bamboo (Phyllostachys edulis) and Phoebe chekiangensis and created a physical barrier to the root system without blocking chemical communication using four treatments: mixed planting with root segregation (MT), mixed planting without root segregation (MS), pure Moso bamboo with root segregation (BT), and pure Moso bamboo without root segregation (BS). We investigated changes in soil and Moso bamboo nutrient content, soil enzyme activity, and microbial metabolic limitation. The results show that mixed planting and root segregation significantly affected soil and plant nutrient content and soil enzyme activities. Compared to the two pure Moso bamboo treatments, mixed planting increased microbial carbon limitation but decreased microbial nitrogen limitation. Physical segregation between roots increased microbial carbon use efficiency (CUE) compared to no segregation. Random forest analyses revealed that the best predictors of soil C and N limitations and CUE were microbial biomass and dissolved organic nitrogen (DON), respectively. Partial least squares path modeling indicated that mixed planting and root separation, directly and indirectly, affected soil microbial metabolic limitation through their effects on soil nutrients, microbial biomass, and enzyme activities. Carbon limitation significantly increased plant nutrient contents. Our study provides further insights into factors influencing nutrient limitation, CUE, and plant nutrient allocation strategies in mixed Moso bamboo plantations.