During the fermentation of milk with lactic acid bacteria, biologically active sequences, known as bioactive peptides, can be produced. Among the most important biological activities attributed to these peptides are metal-chelating (mainly iron and calcium) and ACE-inhibitory activities. Three probiotic strains (Lactobacillus plantarum, Lb. pentosus and Lb. acidipiscis) were isolated from double cream cheese produced in Chiapas (Mexico). This study aims to assess the metal-chelating (iron and calcium) and ACE-inhibitory activity during the milk fermentation by each microorganism and by a mixed culture. Fermentations were performed using reconstituted skim milk powder. Milks were inoculated with 2% of each of the microorganisms or mixed culture. Fermentations were performed at 37°C for 48 h. Samples were taken every 8 h for measurement of microbial growth, protein concentration, degree of proteolysis (TNBS method), metal-chelating (calcium and iron) and ACE-inhibitory activity. Variations on free amino group concentration with respect to the initial concentration that could indicates proteolysis during fermentation were observed. The initial iron-chelating activity was high (68 ± 2%), and was attributed to the fact that some milk proteins have the ability to bind iron. However, during the fermentation the iron-chelating activity increased (99 ± 0.6% of iron-chelating activity in Lb. plantarum fermentation). The other fermentation batches showed a lower percentage of iron-chelating activity. In the case of the calcium-chelating activity, an initial activity of 5 μM Ca²⁺/mg protein which increased up to 18 μM Ca²⁺/mg protein in Lb. acidipiscis, Lb. plantarum and the mixed fermentations could be measured. Lactobacillus plantarum fermentation showed the lowest ACE inhibitory activity (its maximum value is 78 ± 2.3% of ACE-inhibition) while other fermentations reached 97 ± 3% of ACE-inhibition activity. The initial ACE-inhibition activity was 35 ± 3%. All the fermentation batches performed generated metal-chelating (calcium and iron) and ACE-inhibitory activities in these in vitro tests which could be important for the production of functional dairy products.