Artemisinin, being extracted from Artemisia annua L., is recommended by the World Health Organization (WHO) as a drug for the initial treatment of malaria. During the growth and development of Artemisia annua L. in field cultivation, a large amount of this anti-malarial compound could be released into soil ecosystems by rain leaching, root exudation and plant residues, inhibiting the growth of plants around Artemisia annua L. and microbial reproduction in soils. Ectomycorrhizal fungi (ECMF) can increase the growth, nutrient uptake and stress resistance of woody plants following the formation of symbiotic associations, which is important for the sustainability of forest ecosystems. The allelopathic effects of this anti-malarial compound have been seldom reported on ECMFs and plants. In the present experiment, two ECMF strains, Suillus luteus 8 (Sl 8) and Lactarius delicious3 (Ld 3), were isolated in the pine forest in Chongqing, China. They were grown in Pachlewski liquid medium to study the effects of different concentrations of artemisinin (0, 20, 40, 80 mg/L) on fungal growth, efflux of H⁺ and organic acids, and uptake of nitrogen, phosphorus and potassium in vitro. Our results showed that the growth of the two ectomycorrhizal species was significantly inhibited by artemisinin. After being cultured for 14 days in the dark, fungal biomass was decreased by 89.13%(Sl 8)and 26.89% (Ld 3), respectively compared to the control (without artemisinin), indicating the higher sensitivity of Sl 8 than Ld 3 in response to artemisinin. Oxalic acid, succinic acid, malic acid, and citric acid were detected in the culture mediums with Ld 3 inoculated, while only two organic acids, oxalic acid and citric acid, were found with Sl 8. As artemisinin concentrations increased, the efflux of both H⁺ and organic acids from Ld 3 were increased in contrast to Sl 8 which had a low accumulation of H⁺ and oxalic acid but high citric acid in the culture medium. In addition, uptake and concentration of N, P and K in the fungal hyphae were significantly reduced following the addition of artemisinin. The capability of nutrient absorption by Sl 8 was almost lost under 80 mg artemisinin/L. Overall, our results showed that the two ECMFs varied significantly in H⁺ efflux, organic acid accumulation, growth suppression and nutrient uptake inhibition under artemisinin treatment. The results further suggest that artemisinin could alter the growth and nutrient uptake of ECMFs in forest soils, which could potentially risk changes in the distribution, performance and biodiversity of ECMFs in forest ecosystems. Artemisinin decomposes slowly, potentially leading to large amounts of the compound remaining in soils following the cultivation of Artemisia annua L. on a commercial scale. Artemisinin in those soils, particularly in Three Gorges areas in China where a large amount of Artemisia annua L. are grown, could influence not only the biodiversity of ECMF species directly in forest ecosystems but also the growth and health of forests indirectly. Improved understanding of the allelopathic effects of artemisinin on ECMFs could maintain the fungal biodiversity and function in sustainable forests, which is important for the health of natural forests.