In the present work, the nejayote (nixtamalization residue from maize) as a culture medium for probiotic bacteria and production of bacteriocins was evaluated. Nejayotes obtained from three mills were characterized: reducing sugars 0.166-0.818 g/L, total sugars 23.57-63.41 g/L and protein 5.66-12.92 g/L. The fermentation was performed at 37°C for 48 h by: Lactobacillus casei IMAU60214, Lactobacillus rhamnosus GG and Lactobacillus helveticus IMAU70129. Consumption of reducing sugars from 60-65%, total sugars from 6-30% and protein from 1-15% were observed. Furthermore, bacteriocin production was evaluated with the supernatants of fermentation. The greatest inhibition by the presence of bacteriocins was obtained with the supernatants of fermentation of Lactobacillus casei IMAU60214, and Lactobacillus helveticus IMAU70129. The highest inhibition was 14% for Listeria innocua ATCC33090 and 10% for Escherichia coli K-12. Bacteriocins presence was confirmed with addition of proteinase K. Since bacteriocins produced inhibited to L. innocua (Gram +) and E. coli (Gram -) can be said that they probably have a wide spectrum of inhibition. In conclusion, nejayote can be used for the production of probiotics and bacteriocins but need further investigation to optimize the production of bacteriocins.