Introduction: We sought to assess the knowledge, attitudes, anxiety, and preventative behaviors of healthcare workers among high-risk persons regarding Covid-19 affliction. Methodology: The participants in this cross-sectional study were healthcare workers (physicians, nurses, administrators, pharmacists, medical lab technologists, non-physician specialists, and technicians) working in various hospitals and clinics in Pakistan’s twin cities (Islamabad/ Rawalpindi). To assess knowledge, attitude, anxiety, and preventive behaviors about Covid-19 affliction, a self-administered questionnaire was employed. Results: Every participant (n=470) said they were aware of the Covid-19 affliction. Regarding the knowledge of HCWs, 65.5% have good knowledge with a mean score of 6.77±1.17. It was discovered that physicians and pharmacists scored much higher on knowledge tests than other health professionals (P< 0.0001). The percentage of positive attitudes among healthcare workers and the mean score was 93.4 and, 7.22±1.24, associated with education, i.e. P = 0.045. 16.1% of HCWs reported having mild anxiety, 57.9% had moderate anxiety, and 26% had severe anxiety with a mean score of 12.29±3.73 with a significant association with education (P<0.0001), healthcare domain (P<0.0001), and frontline HCW domain (P<0.0001). The preventive behaviors indicate that 93.8% had good conduct (mean score = 3.65±0.63) with a positive association with education (P=0.045) and the healthcare domain (P=0.045). The Healthcare workers exhibited adequate awareness regarding Covid-19 affliction. Conclusion: The knowledge, attitudes, anxiety, and preventative behaviors of HCWs in twin cities of Pakistan related to the Covid-19 affliction study are relatively positive; however, anxiety problems in the present research must be resolved to effectively counter the Covid-19 affliction.