•The protein fractions of the Andean seeds of kanihua variety Ramis and Cupi-Sayhua have a high content of proteins and important essential amino acids.•The antimicrobial hydrolysates of Alcalase and Pepsin-pancreatin sequential system of the kanihua protein fractions presented different degrees of hydrolysis and are novel nutraceutical ingredients in food design and bio-preservatives of food origin.•28 antimicrobial hydrolysates of the kanihua protein fractions presented more significant antimicrobial activity with the enzyme:substrate ratio (1:10), the protein fraction glutelins and the microorganism E. coli, followed by S. aureus, and finally C. albicans.•Four AMPs of kanihua protein fractions purified by chromatography showed significant PI against E. coli, S. aureus, and C. albicans. Three AMPs of globulins and glutelins presented cationic and anionic charge, and one Glutelins gave anionic charge with the MIC of E. coli was 95%. The kanihua (Chenopodium pallidicaule Aellen) Andean grain from the Peruvian Altiplano presents proteins of 15% to 19%. The objective was to obtain purified bioactive antimicrobial peptides (AMPs), hydrolyzed with Alcalase and Pepsin-pancreatin sequential system of protein fractions of kanihua varieties Ramis (KR) and Cupi-Sayhua (KS), and hydrolysates with different degrees of hydrolysis (DH) and percentage inhibition (IP) of the growth of E. coli, S. aureus, and C. albicans. To obtain AMPs, nutraceuticals, bio-preservatives, and novel ingredients in food design. The results showed 216 hydrolysates (1%, w/v), only 28 presented significant difference compared to controls (IP ≥ 45%, p ≤ 0.05), 4 AMPs were purified by chromatography, glutelins KS 4 h (1:10) stood out with DH 40% and IP 52% and 70% of S. aureus and C. albicans, respectively (p ≤ 0.05), showed minimum inhibitory concentration (MIC) of 95% for E. coli (p ≤ 0.05), and presented an anionic charge. In conclusion, the simulated digestion in vitro showed higher DH (7%–67%) than Alcalase (13%–54%); the majority were extensive; of 28 hydrolysates with IP ≥ 45% 4 AMPs with important IPs were obtained, and one was anionic.