Human milk constitutes the best food that can be given exclusively to the newborn, generating the best and most important physical-biological link between the mother and her newborn. We conducted an observational descriptive and cross-sectional retrospective study on the physicochemical properties and microbiological quality of human milk supplied by 68 lactating mothers between 15 and 39 years of age from two Colombian socioeconomic strata in the Tolima department. We evaluated type of milk colustrum transition and mature, with respect to the quality parameters established in human milk bank programs, in addition to Chloride, phenol, phosphorus content and protein profiles for using electrophoresis. Microbiological analysis discards excessive loads of lactose positive Enterobacter in terms of MPN, showing values suitable for the consumption of infants. The electrophoresis technique reveals the presence of lactalbumin, immunoglobulin, casein, albumin and lactoferrin, not always reported in this type of work. This research can be a guide and initiative for the development of similar work in other areas of the country and with it the strengthening of global breastfeeding programs.