Background: Effective infection control in operation theatres (OTs) depends heavily on nurses' knowledge of, and adherence to, aseptic techniques. At Hospital Queen Elizabeth II (QEH II), Sabah, internal audits documented a rise in OT nosocomial infection rates from 4.0% in 2020 to 5.1% in 2023, alongside a hand hygiene compliance rate of only 70%, raising concern about the adequacy of current infection control practice. Objective: This study evaluated the level of knowledge and practice of aseptic techniques among nurses working in the OTs of QEH II and examined the relationship between these two variables. Methods: A quantitative, cross-sectional design was employed. Using Krejcie and Morgan's (1970) formula applied to a population of 180 OT nurses, a stratified random sample of 95 respondents was proportionally recruited across the morning, afternoon, and night shifts. Knowledge was measured with a 20-item multiple-choice instrument adapted from the Aseptic Techniques Knowledge Scale (Lee, 2017; Cronbach's α = 0.85), and practice was measured with a 20-item, 3-point Likert-scale instrument adapted from the Aseptic Technique Compliance scale (Smith, 2019; Cronbach's α = 0.82). Data were analysed descriptively (frequency, percentage, mean, standard deviation) and inferentially using the chi-square test of independence, following confirmation of non-normal distribution via the Shapiro-Wilk test. Results: Nearly half of the participants (49.5%; mean = 14.25, SD = 1.75) demonstrated moderate knowledge, while 30.5% showed low knowledge and 20.0% showed high knowledge. For practice, 55.8% (mean = 20.75, SD = 3.15) demonstrated moderate adherence, 25.3% demonstrated poor adherence, and 18.9% demonstrated good adherence. A chi-square test of independence revealed a statistically significant association between knowledge and practice levels, χ² (4, N = 95) = 42.67, p < .001. Conclusion: Knowledge and practice of aseptic techniques among OT nurses at QEH II were predominantly moderate, and higher knowledge was significantly associated with better practice. However, systemic barriers, including workload pressure, staffing shortages, and inconsistent resource supply, appeared to constrain the translation of knowledge into consistent practice. Targeted competency-based education, simulation-based training, and organisational-level investment in staffing and supplies are recommended to close the knowledge-practice gap and reduce nosocomial infection risk in surgical settings.