Background/Aims Nosocomial infections are transmitted in hospitals and can be air- or vector borne, through direct or indirect transmission. Medical appliances and surfaces serve as reservoirs for micro-organisms that cause nosocomial infections. This study aimed to assess midwives' attitudes and self-reported practice of nosocomial infection prevention in Olabisi Onabanjo University Teaching Hospital, Nigeria. Methods A cross-sectional descriptive study was conducted to investigate midwives' attitudes to and practice of prevention of nosocomial infections in a sample of 107 respondents. Data were collected with a structured pre-tested questionnaire. Attitude was measured using a 5-point Likert scale to rate agreement with seven statements (maximum score: 35) while practice was measured using a 4-point scale against eight statements (maximum score: 32). The data were analysed using descriptive and inferential statistics, with the Chi-squared test used to assess associations between characteristics and adequacy of practice. Results The majority of midwives (84.1%) had a positive attitude to prevention of nosocomial infections, with a mean score of 3.42±0.16. However, 51.4% had inadequate practice of prevention, with a mean score of 3.84±3.22. A significant association was found between education and practice of nosocomial infection prevention (P<0.001), and experience and practice (P<0.001). Conclusions While midwives' attitudes to nosocomial infections were good, their practice was below average. This highlights the need for efforts to ensure compliance with standard precautions.