Although several studies have been conducted to assess dentists' knowledge of and attitudes towards human immunodeficiency virus (HIV)/acquired immune-deficiency syndrome (AIDS), few have targeted dental assistants. The main aims of this study were to assess the knowledge of and attitudes towards HIV/AIDS among dental assistants in Kuwait and to compare the knowledge and attitudes of dental assistants at Kuwait University Dental Center (KUDC) with those of dental assistants in Ministry of Health (MoH) hospitals. The secondary objective was to determine if any intervention was needed to provide more information to dental assistants on HIV/AIDS. A cross-sectional study was conducted by distributing questionnaires. The study sample included 85 dental assistants from each of KUDC and the MoH. The questionnaire included questions to assess the assistants' knowledge and attitude towards HIV/AIDS. Statistical data analysis was conducted using SPSS 20.0. Qualitative data were analysed using the Pearson chi-square text for any association or the Z-test for proportion to test the significance of differences. A total of 167 questionnaires were completed, returned and analysed. KUDC dental assistants were found to have significantly more knowledge about HIV/AIDS than their MoH counterparts, whereas the assistants at the MoH clinics displayed a more positive attitude towards patients with HIV/AIDS (P < 0.05). Although dental assistants at KUDC were more knowledgeable than those at the MoH clinics, there are still some misconceptions that need to be addressed, in addition to the negative attitudes displayed by some of the respondents. It would therefore be beneficial to increase awareness about HIV/AIDS patients through lectures, seminars and workshops targeting dental assistants.