This study describes an α-lactalbumin folding variant from human milk with bactericidal activity against antibiotic-resistant and -susceptible strains of Streptococcus pneumoniae. The active complex precipitated with the casein fraction at pH 4.6 and was purified from casein by a combination of anion exchange and gel chromatography. Unlike other casein components, the active complex was retained on the ion-exchange matrix and eluted only with high salt. The eluted fraction showed N-terminal and mass spectrometric identity with human milk α-lactalbumin, but native α-lactalbumin had no bactericidal effect. Spectroscopic analysis demonstrated that the active form of the molecule was in a different folding state, with secondary structure identical to α-lactalbumin from human milk whey, but fluctuating tertiary structure. Native α-lactalbumin could be converted to the active bactericidal form by ion-exchange chromatography in the presence of a cofactor from human milk casein, characterized as a C18:1 fatty acid. Analysis of the antibacterial spectrum showed selectivity for streptococci; Gram-negative and other Gram-positive bacteria were resistant. The folding variant of α-lactalbumin is a new example of naturally occurring molecules with antimicrobial activity.