Abstract Background Transmission of HIV in South Africa continues to be high due to a large proportion of individuals living with undiagnosed HIV. Uptake of HIV testing is influenced by a multitude of factors including the patient’s knowledge and beliefs about HIV. Methods This study sought to quantify the impact of knowledge and attitudes on HIV testing acceptance in an emergency department by co-administering a validated HIV knowledge and attitudes survey to patients who were subsequently offered HIV testing. Results During the study period 223 patients were interviewed and offered HIV testing. Individuals reporting more negative overall attitudes (p = 0.006), higher levels of stigma to HIV testing (p