Background <p>A key concern for global public health is nosocomial infections. Essential to the fight against nosocomial infection, is healthcare professionals’ knowledge and attitudes. Therefore, this study investigated healthcare professionals’ knowledge and attitudes toward nosocomial infection at the Kiruddu Referral Hospital, Kampala, Uganda.</p> Methods <p>A facility-based cross-sectional study was carried out at Kiruddu Referral Hospital in Kampala, Uganda. We selected the participants using simple random sampling. Data were collected from a total of 78 healthcare personnel using pretested, structured, self-administered questionnaires. We used SPSS version 20.0 for data analysis and applied descriptive statistics to present the frequencies and percentages. Pearson’s Chi-square test was used to evaluate the association between independent factors and knowledge and attitude (KA) ratings on hospital-acquired infection (HAI) prevention. P-values less than 0.05 were regarded as statistically significant.</p> Results <p>Among the different categories of health workers, doctors exhibited the highest level of knowledge. There was a significant association between knowledge scores and occupation (χ²LR = 25.610; <i>P</i> = 0.000). The mean knowledge scores across different infection prevention aspects were as follows: hand hygiene (82.2 ± 18.9), PPE use (71.8 ± 23.1), sharp disposal and sharp injuries (59.2 ± 25.7), and waste management (57.4 ± 29.9). Notably, 20.5% of participants did not change PPE between patients, and 44.9% indicated that their workload negatively impacted their ability to follow infection prevention standards.</p> Conclusion <p>The study highlighted gaps in healthcare personnel’s knowledge and attitudes toward infection prevention. It is therefore important to provide regular targeted training programs emphasizing underrepresented areas, PPE availability, strengthen policy enforcement, and integrate infection prevention education into medical and nursing curricula.</p>