[Background] Bacillus velezensis HN-Q-8 has biocontrol effect on Rhizoctonia solani effectively. [Objective] Identify the inhibition substances produced by Bacillus velezensis HN-Q-8. [Methods] Both effects of strain HN-Q-8 on five pathogens of strain potato and stability of fermentation broth were detected though the growth rate method. Active substances of HN-Q-8 were identified using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). [Results] The fermentation broth of strain HN-Q-8 exhibited strong inhibitory activity against Rhizoctonia solani, Fusarium oxysporum, Alternari solani and Phytophthora infestans, with the inhibition rates ranging from 50% to 90%. The inhibition rates of fermentation broth retained 74% after 35 min exposure to UV, and 92% after a 10 hour exposure to natural light, respectively. The fermentation broth retained 98% antifungal activity after thermal treatment at 100 °C. In addition, the antifungal activity of fermentation broth was not affected by trypsin and protease K. However, the fermentation broth could not endure both extreme acid and alkali treaments, and the suitable pH ranged from 4.0 to 10.0. The methanol extract from strain HN-Q-8 had significant inhibition effect on Rhizoctonia solani, with the two main active substances in it including fengycin and surfactin. [Conclusion] The fermentation broth of strain HN-Q-8 exhibited stability and strong antifungal activity, and has promising prospects for its further study, development and application.