In the Era of COVID-19 outbreak, hand hygiene become a particularly critical for frontline healthcare workers (HCWs) who are overstretched and for whom this key routine task must be easy to complete and effective to prevent Healthcare-associated infections (HAIs). Therefore, this current study aims to assess the level of knowledge, attitude and practice of hand hygiene among the HCWs and the potential factors influencing hand hygiene (HH) compliance according to the hand hygiene protocols at primary health care (PHC) centers in Prince Sultan Military Medical City (PSMMC), Riyadh, Saudi Arabia. This cross-sectional study included clinical staff working at PHC centers. A pre-validate questionnaire was used with observational approach of the application of HH among the study sample. The data were analyzed using SPSS software, and descriptive statistics and logistic regression models were produced. A P-value less than 0.05 with 95% confidence interval was used to declare statistical significance.Of 425 clinical study participants, 263 (61.9%) were females, 169 (39.8%) were at age group 31-39 years, and 215 (50.6%) were nurses/laboratory specialist, and 281 (33.9%) were from departments of high risk of contamination. Overall, the average knowledge score was 75%. However, there were significant higher scores in knowledge level among nurse and laboratory specialist had than the rest (P < 0.01) and three times likely more positive practice of hand hygiene in comparison to others [Adjusted Odds Ratio (AOR) = 3.285, 95% CI, 1.764-6.116].Health care workers in this study showed a good score of hand hygiene knowledge but suboptimal practices which could be addressed the need for multimodal training program on strict implementation of the guidelines and compliance with hand hygiene best practices.