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Background: Healthcare-associated infections (HAIs) are a significant threat in healthcare settings. Since nurses have the most day-to-day contact with patients, their knowledge about infection control (IC) practices is crucial in preventing HAIs. We therefore conducted a study to assess the knowledge and awareness of IC practices amongst nurses across hospitals in India., Methods: An online survey-based, cross-sectional, descriptive study for nurses was conducted in July-August 2021, through a multiple-choice questionnaire, administered via a web-based link across 13 hospitals from various cities of India. Five different aspects of IC knowledge were assessed including general IC, standard precautions, transmission-based precautions, bundle care knowledge, and COVID-19 related knowledge., Results: Complete data filled by 1,000 nurses was analyzed. The knowledge of nurses varied across different aspects of IC. A statistically significant association was found between the IC knowledge and the years of experience (P = .003) and the area of working (critical vs semi-critical areas) (P < .001) of nurses. A statistically significant difference was also found in the knowledge of nurses from different hospitals depending upon the accreditation (P < .001) and the teaching status (P = .035), but no significant difference based on the city category of hospital (P > .05). Accreditation showed the strongest association {β = 2.499 (95% CI = 1.67-3.32)} while non-teaching status had a negative impact {β = -1.76 (95% CI = 2.543 to -2.543)} on knowledge using multivariate linear regression analysis., Conclusions: Infection prevention and control is the biggest challenge in any hospital and improving the knowledge and awareness of the nurses on the same is fundamental to its success. A multifaceted approach of continuing education programs, training, and feedback should be undertaken towards improving the awareness and compliance to IC practices., (Copyright © 2022 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)