A blood donor questionnaire and declaration, with deferral of potential donors at a higher risk of blood-borne infections, was introduced in Australia in the mid-1980s to reduce the risk of donation of HIV-infected blood. However, the absolute risk of HIV transmission through blood donation from high-HIV-risk donors has not been estimated. This study presents a new method of assessing the risk posed to the blood supply by selected HIV risk behaviours. A model was developed to estimate the probability of blood donation during the window period for HIV infection. Five scenarios for blood donors were considered: (1) men who have sex with men (MSM), (2) men who have sex with women in Australia, (3) women who have sex with partners from countries with a high HIV prevalence, (4) men who have sex with commercial sex workers in Australia and (5) people injecting drugs used once in a year. Those estimated to be at highest risk of becoming infected and donating in the window period were MSM. Women who have sex with men from countries of high HIV prevalence are at greater risk than men who have sex with female sex workers from Australia. These three groups under current Australian guidelines are deferred from donating blood for 12 months. In Australia, a single episode of injecting drug use is associated with very low risk of HIV transmission. The model presented in this study can be used to assess the impact of selected individual risk behaviours on the safety of the blood supply.