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The use of donated expressed breast milk (DBM) is encouraged in South Africa, with donor milk banks and legislated policies and programmes available in hospitals serving populations with a high HIV prevalence. Concerns over poor attitudes amongst mothers towards DBM seem to persist. A cross-sectional mixed-methods approach was used in a cohort of mothers of neonates in a regional hospital. Between April 2021 and January 2022, survey data on knowledge, awareness and attitudes towards DBM was collected. Qualitative data from open-ended questions were thematically analysed. Comparative analysis using independent sample t-tests and logistic regression to determine differences in variables and to understand associations related to knowledge was conducted. A total of 163 mothers with a mean (SD) age of 27.1 ± 6.3 years were included; 49.7% of mothers had post-high-school qualifications, 82.8% were unemployed and 87.0% received social security. Some 64.4% had inadequate knowledge of DBM. Awareness of DBM had the strongest association with better knowledge concerning DBM and AdjOR (25.25 95% CI 10.60–68.40; p < 0.001). Negative attitudes towards DBM were largely driven by a lack of knowledge regarding human immunodeficiency virus (HIV) screening of donor mothers and fears of HIV transmission when using DBM. Lack of awareness concerning DBM was associated with poor knowledge of DBM, which may drive poor uptake of this critical nutritional source for at-risk neonates. Implementing targeted awareness programmes on DBM, which begin in the antenatal period and specifically address HIV screening of donor mothers, could address the poor uptake of DBM. [ABSTRACT FROM AUTHOR]