De novo root regeneration (DNRR) is a developmental process that regenerates adventitious roots from wounded tissues. Phytohormone signaling pathways involved in microbial resistance are mobilized after cutting and influence de novo root regeneration. Microbes may positively or negatively influence the development and stress responses of a plant. However, most studies on the molecular mechanisms of de novo organogenesis are performed in aseptic conditions. Thus, the potential crosstalk between organ regeneration and biotic stresses is underexplored. Here, we report the development of a versatile experimental system to study the impact of microbes on DNRR. Using this system, we found that bacteria inhibited root regeneration by activation of, but not limited to, pathogen-associated molecular pattern (PAMP)-triggered immunity. Sensing bacteria-derived flagellin 22 peptide (flg22) inhibited root regeneration by interfering with the formation of an auxin maximum at the wound site. This inhibition relies on the receptor complex that recognizes microbial patterns but may bypass the requirement of salicylic acid signaling.