Staff practice, driven by knowledge, plays a decisive role in hospital infection control. This study aimed to assess and compare knowledge and self-reported practices of infection control among various occupational groups in a rural and an urban hospital in Vietnam. Questionnaires consisting of items on knowledge and practices were collected from 339 hospital staff with varying occupations. For analysis, total knowledge or practice score ranged from 0-15. Mood's median test was performed to compare median scores. Post-hoc analysis of ordinal logistic regression models was applied to test differences in scores among occupational groups. The majority of hospital staff had good or adequate knowledge (median score: rural = 11.8; urban = 12), but the score range was wide (1.4-14.5). Self-reported practices in the urban hospital were likely to be better than in the rural one (p = 0.003). Self-reported practices yet not completely satisfactory, indicating the need for continuing professional development in both settings. Overall, cleaners had lower scores than both physicians and nurses, highlighting the need for tailored education in this topic. Future infection control strategies within the hospitals might want to assess the difference between the staff's self-reported practice and their actual real practice. These findings can be of value in many other similar settings.