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.