The ammonia oxidation process is the rate-limiting step in soil nitrification, driven by ammonia-oxidizing microorganisms, and plays a critical role in the soil nitrogen cycle. While the underlying mechanisms remain unclear, previous studies have shown that the application of biochar can effectively reduce the NH4+-N content in continuous cropping soil of Panax notoginseng, thereby alleviating continuous cropping obstacles. This study conducted an indoor soil cultivation experiment to investigate the effects of rice-husk biochar and rubber-wood biochar on the physicochemical properties and ammonia-oxidizing microbial communities of P. notoginseng. The results indicated that the addition of both rice husk biochar and rubber wood biochar increased soil pH and the content of available K, significantly promoted soil nitrification, and greatly reduced the NH4+-N content in the soil. While biochar application notably affected the composition and diversity of ammonia-oxidizing bacteria (AOB) communities, the impact on the soil ammonia-oxidizing archaea (AOA) communities was relatively limited. Biochar application significantly increased the relative abundance of Nitrosospira while decreasing the relative abundance of Nitrosovibrio. Additionally, the relative abundance (RA) of Nitrosospira showed a significant positive correlation with soil net nitrification rate and a negative correlation with NH4+-N content (P<0.05). Plate culture experiments indicated that when the NH4+-N content was below 10 mg/L, the growth of Fusarium oxysporum, a key root rot pathogen of P. notoginseng, was significantly promoted with the increase in the NH4+-N content in culture medium. Therefore, biochar application can effectively regulate the soil physicochemical properties and AOB communities, promote soil nitrification, reduce the accumulation of NH4+-N in the soil, and alleviate continuous cropping obstacle of P. notoginseng caused by soilborne disease.