This study aimed at identifying the level of HIV-related discriminatory attitudes and related factors in a purposively-selected sample of healthcare workers (HCWs) in Bangladesh. In total, 526 HCWs from a number of hospitals and healthcare centres were interviewed using a structured questionnaire. A moderate level of discriminatory attitudes was observed. The factors associated with a high level of such attitudes among the HCWs were: high level of irrational fear about HIV and AIDS; working in teaching hospital rather than in non-teaching hospital and diagnostic centres; low level of education; and being male. The results indicate that programmes to reduce irrational fear about transmission of HIV are urgently needed.