Nosocomial infections are among the major challenges faced by health systems worldwide, with hand hygiene being one of the simplest and most important preventive measures. This study aimed to assess the effectiveness of an educational intervention based on the Theory of Planned Behaviour (TPB) in improving hand hygiene behaviour among hospital nursing staff. A total of 194 nursing staff members were selected using a random sampling method and randomly assigned to intervention and control groups. Data were collected through researcher-made questionnaire based on the TPB and the World Health Organization’s five moments of hand hygiene. The intervention programme was developed and implemented following a pretest, response analysis, and educational needs assessment. The results showed a significant change in the mean scores of subjective norms, perceived behavioural control, behavioural intention, and behaviour at baseline, immediately after, and 2 months after the intervention in the intervention group. Hand hygiene adherence at the moments of ‘before touching a patient’, ‘after body fluid exposure’, and ‘after touching a patient’ showed significant differences in the intervention group. Given the effectiveness of TPB-based intervention and the influence of factors like the environment and reinforcement, combining TPB with constructs from other behaviour change theories, particularly Social Cognitive Theory is recommended to improve hand hygiene compliance.