Introduction: With the potential threat of an avian influenza (AI) pandemic, healthcare workers (HCWs) are expected to play important roles, and they encounter significant stress levels from an expected increase in workload. We compared the concerns, perceived impact and preparedness for an AI pandemic between HCWs working in public primary care clinics and a tertiary healthcare setting. Materials and Methods: An anonymous, self-administered questionnaire was given to 2459 HCWs working at 18 public polyclinics (PCs) and a tertiary hospital (TH) in Singapore from March to June 2006. The questionnaire assessed work-related and non-work-related concerns, perceived impact on personal life and work as well as workplace preparedness. Results: We obtained responses from 986 PC and 873 TH HCWs (response rate: 74.6% and 76.7%). The majority in both groups were concerned about the high AI risk from their occupation (82.7%) and falling ill with AI (75.9%). 71.9% accepted the risk but 25.5% felt that they should not be looking after AI patients with 15.0% consider resigning. HCWs also felt that people would avoid them (63.5%) and their families (54.1%) during a pandemic. The majority expected an increased workload and to feel more stressed at work. For preparedness, 74.2% felt personally prepared and 83.7% felt that their workplaces were prepared for an outbreak. TH HCWs were more likely to be involved in infection-control activities but the perception of infection-control preparedness in both groups was high (>80.0%). Conclusions: HCWs in both public primary and tertiary healthcare settings felt prepared, personally and in their workplaces, for a pandemic. Their main concerns were risks of falling ill from exposure and the possibility of social ostracism of themselves and their families. Preparedness levels appeared high in the majority of HCWs. However, concerns of HCWs could affect their overall effectiveness in a pandemic and should be addressed by incorporating strategies to manage them in pandemic planning.