For a study, researchers sought to identify the nutritional content and bioactive substances in milk from long-term lactation to locate new donors’ milk for human milk banks. About 43 moms of term infants term infant human milk (TIHM) (3–6 weeks of lactation) and 50 women who had breastfed for over a year provided human milk samples of long-nursing human milk (LNHM). The milk had to be collected within 24 hours. The fat, protein, carbohydrate, and energy content of human milk were determined using the MIRIS human milk analyzer; lactoferrin and vitamin C content were determined using reversed-phase high-performance liquid chromatography (RP-HPLC); total antioxidant activity (TAC) and lysozyme were determined using an enzyme-linked immunosorbent assay (ELISA), and the activity of glutathione peroxidase (GPx) and catalase (CAT) via spectrophotometric methods. Both sets of moms’ sociodemographic information have been collated. LNHM had a higher fat content and energy value than TIHM. The protein composition of LNHM and TIHM was the same. LNHM has a lower carbohydrate content than TIHM. In comparison to TIHM, LNHM had increased TAC, CAT, and GPx activity. There were no significant variations in lysozyme, lactoferrin, or vitamin C concentration across the groups studied. The results indicate that human milk retains high nutritional value after 12 months of breastfeeding, does not lose bioactive components, and can be used in human milk banks.