Fusarium graminearum is one of the most problematic phytopathogens in US agriculture. This fungus causes head blight, foot rot, and damping off on wheat and barley. The infection lowers the grain yield and causes contamination of the grain product with mycotoxins. Effective control measures are lacking, and new fungicides that kill F. graminearum but remain safe and economical to use are needed. Newly synthesized aminoglycosides (JL22, JL38, JL39, JL40, NEOF004, NEOF005), classic aminoglycosides (amikacin, gentamicin, kanamycin A, kanamycin B, neomycin, and ribostamycin), and a lipopeptide, syringomycin E (SRE), were studied to determine their antifungal potential to control F. graminearum. Aminoglycosides are protein synthesis inhibitors that mainly target bacteria, but a few were recently observed to kill fungi. They consist of an aminocyclitol ring bound with two or more amino sugars. Novel aminoglycosides were recently synthesized using novel glycodiversification synthetic schemes involving the replacement of the original amino sugars with unusual amino sugars. SRE is an antifungal lipodepsinonapeptide produced by Pseudomonas syringae pv. syringae. This bacterium is an opportunistic pathogen in a wide range of plant species and produces several fungicidal lipopeptides. SRE forms pores on fungal plasma membrane and causes ion fluxes. An enhancement of its antifungal activity is reported in the presence of rhamnolipid surfactants. The antifungal activities of various aminoglycosides, SRE, and a SRE-rhamnolipids mixture were determined against F. graminearum by measuring in vitro minimum inhibition concentrations (MICs) and in planta lesion area and chlorosis development using a leaf infection assay protocol. It was determined that using Tween® 20 at 0.2 % (v/v) concentration in the leaf infection assay promotes lesion development by F. graminearum with minimum phytotoxicity. In vitro, SRE, SYRA, and synthetic aminoglycoside JL38 showed the best antifungal activities. With the in planta assay, all three antifungal agents prevented infection by F. graminearum. However, inconsistent phytotoxicities were observed with SRE and SYRA that were influenced by the Tween® 20 surfactant included in the leaf infection assay. How Tween® 20 induces these phytotoxic inconsistencies is not known.