Background: Hospital clinicians report poor psychosocial well-being during the COVID-19 pandemic. Few studies have reported data at more than one time point. Aims: To compare psychosocial well-being among hospital clinicians at two different time points during the COVID-19 pandemic in 2020. Methods: Participants included doctors, nurses, midwives and allied health clinicians at a multi-site, public health service in Melbourne, Australia. Data were collected via two cross-sectional, online surveys: May to June (wave 1; n = 638) and October to December 2020 (wave 2; n = 358). The Depression, Anxiety and Stress Scale (DASS-21) assessed psychological well-being in the past week. Investigator-devised questions assessed COVID-19 concerns and perceived work impacts. General linear models were used to assess impact of wave on psychological distress. Results: There were no significant demographic differences between the two groups. Both positive (e.g. learning experience) and negative (e.g. risk of getting COVID-19) impacts were reported. In both waves, staff were most concerned about health risks to family members. Wave 2 respondents were significantly more likely than wave 1 respondents to indicate concerns about colleagues having COVID-19, increased workloads, leave cancellation and increased conflict at work (all P < 0.001). Adjusting for sex, age, self-rated health and discipline group, depression, anxiety and stress scores were significantly higher for respondents in the second than the first wave (all P < 0.001). Conclusions: Psychological well-being of hospital clinicians was significantly worse during the second wave of the COVID-19 pandemic than the first. Sustained occupational and psychosocial support is recommended even when immediate COVID-19 concerns and impacts resolve.