Alternaria blight, caused by Alternaria alternata, is a recent and serious disease of commercially grown Paulownia trees in Western Australia. This study investigated the effect of temperature, humidity, wounding and leaf age on lesion development. Temperature had a significant effect on infection, with lesion development and expansion observed to increase from 15–25°C, declining between 30 and 37°C. Relative humidity (RH) also influenced infection with limited lesion development observed at ≤92% RH whereas incubation at 98 and 100% RH resulted in large lesions. The optimum conditions determined for lesion development were incubation at 25–30°C with 98–100% RH. Wounded leaf tissue was significantly more susceptible to infection than non-wounded leaf tissue and senescent leaves were more susceptible than newly emerged leaves. Variation in pathogenicity was also observed on senescent leaves infected with different pathotypes of A. alternata. These results appear to correlate well with field observations and may be useful in the control, management and forecast strategies for Alternaria blight in Australian Paulownia plantations.