Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most important pests causing considerable damage to soybean (Glycine max (L.) Merr.) around the world. Biocontrol provides a strategy for sustainable nematode control. Previously, Aspergillus niger NBC001 was isolated from the cysts of Heterodera spp. and able to control H. glycines and promote the growth of soybean in a pot experiment. In this study, the effects of NBC001 on H. glycines density and on the soybean rhizosphere microbial community in a soybean cyst nematode-infested field were studied. The results showed that NBC001 could suppress H. glycines by 31.7％ in the field. High-throughput sequencing analysis showed that NBC001 had no significant influence on soybean rhizosphere microbial community structure, indicating that seed coat-dressing with the concentrated culture filtrate of NBC001 was safe for the soil ecological environment. In addition, high-throughput sequencing results demonstrated that at 10 days post transplantation, NBC001 increased the abundances of Actinobacteria and Acidobacteria, but decreased the abundances of Bacteroidetes and Gemmatimonadetes at the phylum level. Meanwhile, the abundances of Phyllobacterium, Ralstonia and H16 were increased, while the abundances of Adhaeribacter, Gemmatimonas, Sphingomonas, Flavisolibacter were suppressed by application of NBC001. However, at 90 days post transplantation, NBC001 only increased the abundances of Aeromicrobium and RB41 whereas it decreased the abundance of H16. The results indicated that application of NBC001 increased the relative abundances of the beneficial microorganisms such as Actinobacteria, Acidobacteria, Aeromicrobium and Phyllobacterium in the soil. In summary, NBC001 is an eco-friendly biocontrol agent for H. glycines control.