Health care providers including pharmacists are often on the first line when dealing with COVID -19; they can be under threat of contracting and spreading the disease. We aimed to assess and compare their knowledge of hand sanitization during COVID-19 pandemic to improve quality of care. A cross-sectional study was conducted in Jordan, on healthcare providers in different settings from 27 October till 3 December 2020, using a pre-validated electronic questionnaire. Participants (n = 523) were healthcare providers practicing in different settings. Descriptive and association statistical analyses were produced on the data using SPSS 26. Chi square was used for the categorical variables, and One way ANOVA was used on the continuous and categorical variables. A significant difference was recorded in total knowledge mean according to gender (59.78 vs 61.79 p = 0.030) in favor of men, and between pharmacists and other healthcare providers in favor of the latter (59.22 vs 61.45, p = 0.02). No significant difference was generally noticed between those who attended hand hygiene training and those who did not. Healthcare providers' knowledge of hand hygiene was generally good among participants, regardless of training and it was possibly increased because of fear of COVID-19 infection. Physicians were the most knowledgeable in regard of hand hygiene while pharmacists were the least among healthcare providers. Thus, structured, more frequent, and tailored training on hand sanitization in addition to new educational strategies are recommended for healthcare providers, in particular, pharmacists for better quality of care especially in pandemics.