Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative bacterium causing thekiwifruit canker disease, resulting in serious economic losses tothe kiwifruit industry. This study investigated the use of an endophyticfungus, Fusarium tricinctum, obtainedfrom the kiwi plant (Actinidia chinesis) as a potential biocontrol strain against the Psa. F. tricinctum showed an inhibition rate of 59.5%in vitro against Psa. Bioassay-guided isolation was conducted on thecultural broth of F. tricinctum andseven new imidazole alkaloids, (+/-)-fusaritricine J ((+/-)-1) and fusaritricines K-P (2-7), and four enniatins (8-11) wereidentified. Their absolute configurations were established throughextensive spectroscopic methods, quantum chemical calculations, andX-ray single crystal diffraction. Compounds 1, 4, 5, and 8-11 showed comparable anti-bacterial activities against Psa as positivecontrol, with MIC values of 25-50 mu g/mL. Further cellmembrane permeability assay suggested that the most active compound 4 could destroy the bacterial cell wall structure. Hence, F. tricinctum metabolites could be applied as potentialanti-Psa agents, and F. tricinctum couldbe considered a biocontrol strain for the control of the kiwifruitcanker disease.