Objective: Infection poses a significant challenge within the global landscape of healthcare delivery services. It significantly contributes to morbidity and death linked to clinical, diagnostic, and therapeutic interventions. The objective of this study was to evaluate the level of knowledge, awareness, and adherence to infection control protocols among healthcare personnel working in the Intensive Care Unit (ICU) of our hospital. Methodology: The present study employed a cross-sectional descriptive methodology to evaluate the knowledge and hand hygiene behaviors of doctors and nurses, while also identifying factors that influence and either encourage or hinder these practices at Sri Krishna Medical College and Hospital, Muzaffarpur, Bihar. The Knowledge questionnaire consisted of a total of 22 questions, each requiring a true or false response. The knowledge was assessed using a percentage-based grading system. Participants with higher scores demonstrate a greater level of knowledge on hand hygiene. Results: The respondents to the knowledge exam were predominantly male healthcare professionals (HCPs), with 16 (64%) being nurses and midwives. The average age of the participants was 31.4 years, with a standard deviation of 5.8 years. The age range was from 25 to 48 years. Following the completion of bivariate analysis, it was determined that there was no statistically significant correlation between knowledge and several factors including occupation (p = 0.9), gender (p = 0.7), work experience (p = 0.9), age (p = 0.8), and training (p = 0.9). In summary, none of the variables examined exhibited a statistically significant association with the observed disparities in knowledge scores. A total of 44 spot checks were carried out. The availability of water was recorded at 29%, while soap availability was found to be 45%. Conclusion: Healthcare professionals (HCPs) demonstrated a commendable level of understanding on the practice of hand hygiene. The frequency of hand washing increased following exposure to bodily fluids.