Strain Pc3 was isolated from Antarctic seawater and identified as Bacillus amyloliquefaciens. A compound with antifungal activity was purified from the fermentation supernatant of B. amyloliquefaciens Pc3. Its structure was determined to be (S)-2-amino-3-(1H-indol-2-yl) propanoic acid, named as isotryptophan, based on detailed analysis of its nuclear magnetic resonance (NMR) and high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS). Isotryptophan exhibited good thermal stability and antifungal activity against several plant-pathogenic fungi with low minimum inhibitory concentrations. Therefore, the characterisation of isotryptophan from the Antarctic B. amyloliquefaciens Pc3 will facilitate its potential application in the control of plant-pathogenic fungal infection.