Root biomass and length growth, functional traits, and their responses to soil nutrient availability are crucial for resource acquisition under environmental change. Previous studies have focused on the response of root morphological, architectural, and chemical traits to fertilization, while less attention has been given to root biomass and length growth, as well as mycorrhizal symbiosis, impeding a full understanding of root resource acquisition strategies. Here, using a nutrient addition experiment (control, inorganic, and organic nutrients), we explored the responses of function-based root (absorptive fine roots [AFRs] versus transport fine roots [TFRs]) growth, functional traits (morphological, architectural, and chemical traits), and mycorrhizal colonization of C. lanceolat and Q. acutissim, which are the dominant tree species in subtropical China. The results showed that the fine root biomass and total root length of AFRs for both tree species basically decreased significantly after nutrient addition, but TFRs responded less sensitively than AFRs. Nutrient addition significantly increased the mycorrhizal colonization rate in C. lanceolata but decreased it in Q. acutissima. The diameter of AFRs for C. lanceolata increased significantly, while the branch ratio and branch intensity decreased significantly in both inorganic nutrients (NPK) and organic nutrients (F); however, the opposite response pattern occurred for Q. acutissima. Fine root biomass, total root length, and root nutrient concentration exhibited the most plastic responses to changes in nutrient availability. The magnitude of the plastic response of fine root traits was slightly higher in the NPK treatment than in the F treatment. Our findings suggest that the responses of fine root traits and mycorrhizal fungi to nutrient addition were species-specific: C. lanceolata depended on mycorrhizal fungi for resource acquisition, while Q. acutissima could acquire soil nutrient resources by increasing root branching. The contrasting nutrient acquisition strategies between tree species may facilitate plant species coexistence and distribution under soil nutrient change. Fine root biomass and total root length emerged as more pivotal indicators for nutrient acquisition strategies compared to morphological traits.