Background: The increasing prevalence of healthcare-associated infections (HCAIs) represents a significant challenge to patient safety worldwide, particularly in tertiary care settings where the risk of transmission is heightened. Effective infection prevention and control (IPC) practices, including hand hygiene, are pivotal in mitigating these risks. Despite the critical importance of these measures, adherence varies significantly across different healthcare settings, especially in low and middle-income countries. Objective: This study aims to assess the knowledge, attitudes, and self-reported practices related to infection control among healthcare workers in a tertiary care hospital in Larkana, with a specific focus on hand hygiene practices. The study seeks to identify gaps in knowledge and practice that could be targeted to improve overall IPC measures within the hospital. Methods: A cross-sectional survey was conducted among healthcare workers at a tertiary care hospital in Larkana, utilizing a WHO-validated self-administered questionnaire. The survey covered various aspects of IPC, including hand hygiene knowledge, attitudes towards infection control, and self-reported adherence to IPC practices. The participants included doctors, nurses, paramedics, pharmacists, and technicians, with data collected on demographic details, professional roles, and specific IPC practices. Results: Out of 317 respondents, a substantial proportion, 65.93% (209 participants), reported routinely using alcohol-based hand rubs, indicating good practice adherence. Furthermore, 88.32% (280 participants) demonstrated sufficient knowledge on the correct handwashing technique as recommended by the WHO. Comparative analysis within the professional groups revealed that doctors and technicians were more likely to report higher adherence to hand hygiene practices than other healthcare workers. Conclusion: The study underscores a generally high level of knowledge and adherence to hand hygiene practices among healthcare workers in the surveyed tertiary care hospital in Larkana. Despite these positive findings, the variability in self-reported practices across different professional groups suggests the need for targeted educational and policy interventions to ensure uniform IPC compliance.