Abstract: Ethanol losses due to bacterial contamination in fermentation cultures weakens the economics of biofuel production. Lactobacillus species are the predominant contaminant. Bacteriophage lytic enzymes are peptidoglycan hydrolases which degrade Gram positive cell walls when exposed externally and are a novel source of antimicrobials. The streptococcal phage ?SA2 endolysin construct demonstrated strong lytic activity towards 17 of 22 strains of lactobacilli, staphylococci or streptococci maintaining optimal specific activity under fermentation conditions toward L. fermentum substrates. Lactobacillus bacteriophage endolysin constructs LysA, LysA2 and LysgaY showed exolytic activity towards ˜60% of the lactobacilli tested including four L. fermentum isolates from fuel ethanol fermentations. Presence of ethanol (?5%) did not affect lytic activity. Lysins were able to reduce both L. fermentum and L. reuteri contaminants in mock fermentations of corn fiber hydrolysates.