Tree-herb intercropping was proposed for use in remediation of metal(loid)-contaminated soil. Changes in the enzymatic activities and microbial communities in contaminated soil during tree-herb intercropping were studied through dynamic sampling in a greenhouse experiment. Two herb plants, Pteris vittata L. (W) and Arundo donax L. (L), and two tree plants, Morus alba L. (S) and Broussonetia papyrifera L. (G), were selected for tree-herb intercropping, namely SL, GL, GW, SW, and GSLW intercropping. The activities of four enzymes, dehydrogenase activity (DHA), urease activity (UA), sucrase activity (SA) and acid phosphatase activity (APA), are involved in N, C, P cycling and were measured colorimetrically, while the bacterial and arbuscular mycorrhizal (AM) fungal community structures were determined using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that tree-herb intercropping could effectively recover enzymatic activity, and bacterial and AM fungal diversity in metal(loid)-contaminated soil. Compared with treatment without plants, the SA and APA activity after the five tree-herb intercropping treatments were significantly (p < 0.05) increased by 1.64–2.51 times and 2.35–5.70 times after 270 d of cultivation, respectively. Meanwhile, the Shannon-Wiener indexes for bacteria and AM fungi increased by 13.6–17.7% and 20.0–36.9%, respectively. Soil DHA, UA, and SA were slightly affected by tree-herb intercropping; however, APA was further significantly enhanced by SL, GL, and GSLW intercropping when compared to the monoculture treatments after 270 d of cultivation. These results indicated that tree-herb intercropping could effectively mitigate the toxic effects of metal(loid)s on soil enzyme activities and microbial community structures and enhance the potential for ecological remediation of metal(loid)-contaminated soil in mining areas. Display omitted •Tree-herb intercropping improves the biological quality of polluted soil.•Soil enzyme activity and microbial diversity benefited from tree-herb intercropping.•Acid phosphatase activity was further enhanced by SL, GL, and GSLW intercropping.•Tree-herb intercropping had a significant impact on the AM fungal community in soil.