Chitotriosidase is a chitinolytic enzyme expressed by maturing macrophages and preformed in neutrophil granules, suggesting a role in antimicrobial defence. Although available evidence supports a role in anti-fungal immunity, there is a lack of an obvious phenotype in humans homozygous for a mutation which renders chitotriosidase inactive. This may be explained by compensatory effects of enzymes co-expressed with chitotriosidase, such as lysozyme. We have found that chitinase is highly expressed in mouse and human eye, particularly in lacrimal glands. Chitotriosidase is the only member of the chitinase/chilectin gene cluster expressed in the murine eye. As lacrimal glands also produce lysozyme, we have asked whether chitotriosidase, in addition to its documented anti-fungal effects, has synergistic anti-bacterial properties with lysozyme. The effect of recombinant chitotriosidase on the growth of five Gram-positive (Bacillus cereus, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Staphylococcus aureus OatA(+/-)) and two Gram-negative strains (Escherichia coli and Pseudomonas aeruginosa), were tested in a luminometric assay. Recombinant chitotriosidase did not inhibit bacterial growth and did not synergize with lysozyme. Though the expression of chitotriosidase in the eye supports a role in innate immunity, the antimicrobial spectrum appears to be complementary to lysozyme and may indeed be limited to fungi.