A self-administered validated (Cronbach's alpha=0.077) questionnaire was used to assess knowledge, attitude and practice among healthcare workers (HCWs) in Pakistan regarding coronavirus disease 2019 (COVID-19). Findings showed that HCWs have good knowledge (93.2%, N=386), a positive attitude [mean 8.43 (standard deviation 1.78)] and good practice (88.7%, N=367) regarding COVID-19. HCWs perceived that limited infection control material (50.7%, N=210) and poor knowledge regarding transmission (40.6%, N=168) were the major barriers to infection control. Regression analysis indicated that pharmacists were more likely to demonstrate good practice than other HCWs (odds ratio 2.247, 95% confidence interval 1.11–4.55, P=0.025). This study found that HCWs in Pakistan have good knowledge, but there are gaps in specific aspects of knowledge and practice that warrant attention.