Abstract Biochar has been considered an effective approach as soil amendment for decreasing incidences of disease and regulating microbial populations in continuous-cropping soil. Although researches have extensively focused on changes of soil microbes and unbalance of nutrition in continuous-cropping soil, the relationship between soil properties and pathogens by biochar application remains poorly understood. In this study, we applied ITS ribosomal RNA gene profiling to analyze tobacco root microbiota of biochar and non-biochar treatment in a 3-year continuous-cropping tobacco field, comparing firstly planting tobacco as control. We found that biochar application decreased the relative abundance of the soil fungal pathogens (Ceratobasidium and Monosporascus), which are the prime pathogens of tobacco root rot in continuous-cropping soil. Using RDA, co-occurrence and PLS-PM approaches, we provided evidence that there was a negative correlation between fungal genera (especially for Ceratobasidium and Monosporascus) and soil polyphenol oxidase (PPO) activity (R2 incidence rate = − 0.930, R2 disease index = − 0.905, both p