In this study, we investigated the effects of indole on biofilm formation inhibition in Pantoea agglomerans (P. agglomerans). In the biofilm growth assay, indole inhibited biofilm formation across all the growth time. Depending on biofilm growth stage, indole exhibited biofilm inhibition and anti-bacterial effects on planktonic cells. Through the analysis of the proportion rate between biofilm and Colony Forming Units (CFU) and inhibition rate of indole, we confirmed that depending on the biofilm stage of P. agglomerans, indole treatment timing was more important than the treatment duration. By comparing gene expression rates through rt-qPCR P.agglomerans affected by indole was found to significantly change quorum sensing (pagI/R) and indole transportation (bssS) gene expressions. Throughout all, indole exhibited both antimicrobial and anti-biofilm effects on P. agglomerans. In addition, we confirmed the anti-biofilm effects of indole on mature biofilm. In conclusion, indole as a signal molecule, can exhibit anti-biofilm effects through bacterial quorum sensing inhibition and indole affects. Therefore, indole can regulate biofilm bacteria especially gram-negative opportunistic pathogens.