Background Human milk banks play an essential role by providing human milk to infants who would otherwise not be able to receive mother’s milk. Study aim To determine the opinions and attitudes among possible donor mothers regarding human milk banks in one rural region in Bangladesh. Methods A prospective, cross-sectional study following a probability type of stratified cluster sampling technique was used. Included in the study ( N = 121) were mothers aged 20–49 years, with at least one child, who was currently lactating or had breastfed her child, and who resided in the rural community of Bangladesh where the study was conducted. Data were collected through a 21-item, close-ended questionnaire and a face-to-face interview conducted by the researcher at each participant’s home. Results Among the participants, 98.3% ( n = 119) said that they had not heard about human milk banks before speaking with the researchers. Most participants (71.9%, n = 87) said would obtain human milk from milk banks if necessary, but 28% ( n =34) of mothers indicated that they would not receive milk from a milk bank, even if it was necessary for their children. Only 8.3% ( n = 10) said human milk banks were not appropriate according to Islam and 99.2% ( n = 120) did not know about the acceptance of human milk banking practices in Bangladesh. Conclusions For those with religious concerns, a framework for both the donors and recipients can be established. It can be recommended that health education through healthcare personnel (midwives, nurses, gynecologists, pediatricians, and other health professionals) and religious leaders may strengthen the belief and increase awareness among family members about milk banking practices.