In a search for an antifungal substance with activity against the dermatophyte fungus Trichophyton rubrum, strain POC 115 was chosen among different Paenibacillus strains for its phenotypic and genetic characterization and for preliminary characterization of its antimicrobial substance. Strain POC 115 was identified as belonging to Paenibacillus kribbensis. Physico-chemical characterization of the antimicrobial substance showed that it was not stable during heat and organic solvents treatments, but its activity was preserved at a wide range of pH and after treatment with pronase E, trypsin and DNase I. The crude concentrated supernatant of POC 115 culture was partially purified and the fraction presenting antimicrobial activity was further analyzed by UPLC/Mass Spectrometry. Two peaks were observed at 2.02 (mass 1,207 D) and 2.71 (mass 1,014 D) min in the mass chromatogram. The antimicrobial substance produced by POC 115 was correlated to iturin family compounds based on a set of primers designed for the amplification of PKS operon in the POC 115 genome. As happens with the mode of action of the antibiotics of the iturin group, the AMS produced by POC 115 caused the disruption of cytoplasmic membrane of T. rubrum and the subsequent withdraw of the intracellular material. This is the first report on the production of antimicrobial substances in P. kribbensis, and it may be of great relevance as an alternative or supplementary substance to antifungal drugs currently used against dermatophytes.