Breast milk (BM) is a unique food due to its nutritional composition and anti-inflammatory characteristics. Evidence has emerged on the role of Presepsin (PSEP) as a reliable marker of early sepsis diagnosis. In the present study, we aimed to investigate the measurability of PSEP in BM according to different maturation stages (colostrum, C; transition, Tr; and mature milks, Mt) and corrected for delivery mode and gender. We conducted a multicenter prospective case–control study in women who had delivered 22 term (T) and 22 preterm (PT) infants. A total of 44 human milk samples were collected and stored at −80 °C. BM PSEP (pg/mL) levels were measured by using a rapid chemiluminescent enzyme immunoassay. PSEP was detected in all samples analyzed. Higher (p < 0.05) BM PSEP concentrations were observed in the PT compared to the T infants. According to the grade of maturation, higher (p < 0.05) levels of PSEP in C compared to Tr and Mt milks were observed in the whole study population. The BM subtypes’ degrees of maturation were delivery mode and gender dependent. We found that PSEP at high concentrations supports its antimicrobial action both in PT and T infants. These results open the door to further studies investigating the role of PSEP.