Background and Objective: Donor human milk banks are used when breast milk directly from mothers is unavailable or insufficient. Breast milk contains melatonin, which exhibits a 24-hour pattern. Melatonin promotes sleep onset and is barely detected in daytime milk but rises in the evening and peaks early in the morning. Melatonin supports the development of an infant's own circadian rhythm and is important for neurodevelopment. Currently, donor banks pasteurize breast milk using a Holder Pasteurization (HoP) technique where breast milk is treated at a high temperature (+62°) for 30 minutes before cooling to eliminate any pathogens before it is given to infants. It is not known how the pasteurization process affects the melatonin levels in breast milk. The aim of this study was to investigate whether the pasteurization process reduces melatonin levels in breast milk. Materials and Methods: Ten night-time breast milk samples were collected and each divided into two groups; one group remained unpasteurized and the other group was pasteurized using the HoP technique. Results: Melatonin levels between the unpasteurized and pasteurized groups were compared. Results showed that there was a significant reduction after pasteurization (mean ± standard deviation = 51.92 pg/mL ± 19.54 versus 39.66 pg/mL ± 13.05, p = 0.01). Conclusions: It is important to understand that pasteurization can reduce melatonin levels in breast milk because this hormone is considered important to support the neurodevelopment of infants, especially those born preterm. Further focus on the effect of pasteurization techniques on melatonin in donor breast milk is warranted.