The multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex (Nox) is a crucial source off reactive oxygen species (ROS), and plays a significant role in regulating the development and pathogenicity of phytopathogenic fungi. Calonectria ilicicola, a soil-borne phytopathogenic fungus, primarily causes red crown rot (RCR) in soybean, leading to a dramatic decrease in yield and quality during epidemics. In the present study, four Nox enzymes CiNoxA, CiNoxB, CiNoxD and CiNoxR were characterized in C. ilicicola. To investigate the functions of Nox enzymes, we constructed a new PEG-mediated transformation system to target deletion of the CiNox gene. The data showed that all CiNox enzymes were involved in regulating mycelial growth, asexual/sexual reproduction (except for CiNoxB), sensitivity to abiotic stress agents, conidial germination rate, and virulence. Furthermore, disruption of CiNox enzymes significantly weakened their antioxidative abilities. Additionally, nitro blue tetrazolium staining demonstrated that only the single-gene deletion mutant CiNoxB was restricted to superoxide production compared to the other mutants. Collectively, these results suggest that CiNox genes are responsible for modulating the development and virulence of C. ilicicola, while highlighting their potential as novel targets to control the disease caused by C. ilicicola.