Black aspergilli are some of the most common mycotoxigenic fungi in vineyards worldwide. The aims of this research were to assess the occurrence of fumonisin-producing black aspergilli in Australian wine grapes and the effects of environmental factors on fumonisin production by A. niger and A. welwitschiae (syn. A. awamori). Thirty-eight Aspergillus isolates (black aspergilli) were collected from six wine grape varieties grown in Australian vineyards. LC–MS/MS analysis of culture extracts revealed that six isolates produced fumonisins FB2 and FB4. Molecular data revealed that all fumonisin-producing isolates were A. niger and A. welwitschiae. None of the reference isolates, A. carbonarius, A. tubingensis, A. japonicus, and A. foetidus, were positive for fumonisin production. The effects of temperature and water activity on the growth and production of fumonisins were studied using two A. niger and an isolate of A. welwitschiae on synthetic grape juice medium (SGJM) at 20 °C, 25 °C, 30 °C, and 35 °C, and 0.92 aw, 0.95 aw, and 0.98 aw levels. All isolates produced FB2 and FB4 at 0.95 aw and 0.98 aw and 20 °C, 25 °C, and 30 °C. The highest growth rate observed was 14.89 mm/day for A. welwitschiae at 0.98 aw and 35 °C, whereas the highest fumonisin production observed was 25.3 mg/kg at 0.98 aw and 20 °C for A. welwitschiae. None of the isolates produced fumonisins at 35 °C at any water activity levels. To our knowledge, this is the first report on the occurrence of fumonisin-positive isolates of Aspergillus from Australian wine grapes and the impact of the environmental factors on fumonisin production by A. welwitschiae.