Natural elicitors induce plant resistance against a broad spectrum of diseases, and are currently among the most promising biocontrol tools. The present study focuses on the elicitor properties of the cyclic lipopeptide surfactin on wheat, in order to stimulate the defenses of this major crop against the challenging fungal pathogen Zymoseptoria tritici. The protection efficacy of surfactin extracted from the strain Bacillus amyloliquefaciens S499 was investigated through greenhouse trials. Surfactin protected wheat by 70% against Z. tritici, similarly to the chemical reference elicitor Bion^®50WG. In vitro biocidal assays revealed no antifungal activities of surfactin towards the pathogen. A biomolecular RT-qPCR based low-density microarray tool was used to study the relative expression of 23 wheat defense genes. Surfactin significantly induced wheat natural defenses by stimulating both salicylic acid- and jasmonic acid-dependent signaling pathways. Surfactin was successfully tested as an elicitor on the pathosystem wheat–Z. tritici. These results promote further sustainable agricultural practices and the reduction of chemical inputs.