Diabetes mellitus is one of the most common metabolic disorders and a risk factor for bacterial ocular infections. Our aim was to examine the antibacterial activity of tears from patients with diabetes mellitus and diabetic retinopathy. Non-stimulated basal tears were collected from 39 eyes of 35 subjects. The antibacterial activity of tear pools was tested against pathogenic Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 26922 and Pseudomonas aeruginosa ATCC 27853 strains. The levels of 10 antimicrobial and immunomodulatory proteins were analyzed in the individual tear samples of the studied groups by SRM-based targeted mass spectrometry analysis. Disease stage-specific antimicrobial effect was observed in case of Staphylococcus aureus ATCC 29213 strain and a non-disease specific inhibitory effect was observed in case of Pseudomonas aeruginosa ATCC 27853 strain. Changes in the levels of the studied antimicrobial and immunomodulatory proteins in the tears of the studied groups were also observed. The higher ocular infection rate observed in diabetic patients may be the consequence of the decreased antimicrobial activity of tears possibly caused by the changes in the levels of antimicrobial and immunomodulatory proteins.