A cross-sectional survey was conducted to identify the attitudes and behaviours of health care workers (HCWs) in health care settings (HCS) in Fiji involving 369 participants. Self-rated knowledge of HIV and sexually transmissible infections (STIs) varied depending on whether the HCS was divisional or sub-divisional, and varied between the various national divisions. HCWs with experience in HIV, reproductive health and antenatal clinics had higher self-rated HIV knowledge. A high proportion had a fear of catching HIV from HIV-positive clients. This study found high levels of negative attitudes towards clients from vulnerable groups with regards to the transmission and spread of HIV. Study participants also reported observing differential treatment by their colleagues if a client was known to have or was suspected of having HIV. There is a need for further HIV education of HCWs, with training focussed on occupational risk, and on reducing stigma and discrimination of those living with or vulnerable to HIV in Fiji.