Uromycladium acaciae causes a serious rust disease in plantations of non-native Acacia mearnsii (black wattle) in South Africa. Little is known about the biology of U. acaciae, making disease control difficult. Germination studies and artificial inoculations were conducted to identify the optimal environmental conditions for infection by U. acaciae. Germination of teliospores, basidiospores, and urediniospores was assessed at seven temperatures, with or without light. The effect of temperature on infection was also assessed. As was the effect of dew period length on germination of teliospores, production of basidiospores and infection. Teliospores and urediniospores germinated between 5 and 30 °C, with an optimum at 15–25 °C. Basidiospores were produced and germinated at temperatures between 5 and 25 °C, with an optimum at 15–20 °C. The optimum temperature for infection by basidiospores was 15–20 °C. All spore types germinated in 6–24 h under optimal conditions. However, production of basidiospores was severely reduced if teliospore germination was interrupted by dry periods, even if teliospores were re-wetted. Symptoms and telia developed on only one plant exposed to a dew period of less than 12 h, with a dew period of 48 h found to be optimal. Artificial inoculation experiments showed that U. acaciae was only able to infect young, growing tissues. Results of this research can be used to develop an artificial inoculation protocol for resistance screening and in disease risk modelling and forecasting.