Background: Human breast milk is a dynamic and multi-faceted fluid that contains essential nutrients for infant health and development. Its composition changes throughout the stages of lactation, and besides providing the newborns with plenty of nutrients, it also protects them against infections such as sepsis, pneumonia, and enteritis, especially in premature infants. The question of whether breastfeeding is cariogenic or not remains unresolved when comparing the results of different researchers. The objective of this study was to measure the bactericidal effect of breast milk against Streptococcus mutans (S. mutans), which has a high cariogenic potential. Methods: Breast milk samples were collected from 9 donors and divided into 3 groups: Group 1 (fresh milk), Group 2 (milk stored at −18 ◦C for 24 hours) and Group 3 (milk stored at −18 ◦C for 72 hours). From each group, 3 disks soaked in milk were placed on an agar culture of S. mutans. In the same culture a disk soaked with chlorhexidine was placed as a control. Inhibition halos were measured after 24, 48 and 72 hours. Results: Only Group 1 (fresh milk) incubated for 24 hours showed a statistically significant difference, presenting the lowest inhibition (p < 0.05). In the other eight data sets, there was no significant difference showing higher or lower inhibition (p > 0.05). However, there was a trend toward reduced inhibitory capacity when the milk was frozen for 72 hours. Conclusions: Human breast milk showed a bactericidal effect against S. mutans, which has a high cariogenic potential. Given this, we recommend exclusive breastfeeding during the first 6 months of life and thereafter combining breastfeeding and complementary feeding for as long as desired by the child and mother.