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To determine the in vitro antimicrobial activity of N-acetylcysteine (NAC) against common pathogens associated with infectious keratitis in dogs and cats, clinical isolates of <i>Staphylococcus (S.) pseudintermedius</i> (n = 20), <i>Streptococcus (St.) canis</i> (n = 10) and <i>Pseudomonas (P.) aeruginosa</i> (n = 7) of canine and feline infectious ulcerative keratitis and a quality control strain (<i>P. aeruginosa</i> DSM 19880) were tested. The minimal inhibitory concentration (MIC) of NAC concentrations was determined using microdilution methodology. For <i>S. pseudintermedius</i> and <i>P. aeruginosa</i>, NAC concentrations in the range of 1.56 mg/mL (0.156%) to 100 mg/mL (10%), and for <i>St. canis</i>, concentrations ranging from 0.195 mg/mL (0.0195%) to 6.25 mg/mL (0.625%) were tested. For <i>S. pseudintermedius</i>, the MIC was 3.12 mg/mL (0.312%) for all tested isolates. For <i>P. aeruginosa</i> isolates and the quality control strain, the MIC ranged from 3.12 mg/mL (0.312%) to 6.25 mg/mL (0.625%). For <i>St. canis,</i> the MIC ranged from 1.56 mg/mL (0.156%) to 3.12 mg/mL (0.312%). NAC has an in vitro antimicrobial activity against three bacterial species commonly found in infectious keratitis in dogs and cats and therefore may be a promising alternative or adjuvant to topical antibiotics. The results warrant a clinical pilot study to assess the potential of NAC to reduce or replace the use of topical antibiotics in line with the One Health approach.