Several microorganisms are responsible for great economic losses in world agriculture. Preventive and treatment methods are applied to avoid contamination of crops by these microorganisms, however, the use of chemical antimicrobials damages health and the environment. Secondary plant metabolites are safe natural sources of antimicrobials for this application. Fabaceae family has its history described in the literature as a potential source for obtaining antimicrobial bioactive. The objective of this work was to isolate bioactive compounds guided by antimicrobial assays against bacteria and fungi in vitro. Organic extracts were prepared by eluotropic series of leaves of Anadenanthera colubrina var. cebil and were tested against six bacteria and six fungi phytopathogenic. The antimicrobial assays of minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were performed at each purification step that occurred through HPLC-DAD, Flash Chromatography and HPLC-preparative analysis, to confirm the isolation of the bioactive. Through bioguided isolation, the compound p-hydroxybenzoic acid was obtained, which showed activity against the phytobacteria Xanthomonas campestris pv. campestris and Acidovorax citrulli.