Medium-chain fatty acids (MCFAs) and their monoesters were tested for their antibacterial activity against Gram-negative pathogen Aeromonas hydrophila. The antimicrobial effect was evaluated at two temperatures (4 °C and 37 °C) using a standardized method microdilution method in a 96-well microtitration plate. The minimum inhibition concentrations of selected MCFAs were determined as the lowest concentration limiting the growth of A. hydrophila in wells compared to a positive control of ≥ 80%. The results indicated that the most effective compound against A. hydrophila was sucrose monocaprate after incubation at 37 °C (0.625 mg ml–1), whereas monocaprylin was the most effective compound after incubation at 4 °C (1.25 mg ml–1). Free MCFAs showed no antibacterial effects towards this bacterium.. Low solubility and sensory properties could limit the use of fatty acids in aquatic environment, which should be the subject of further studies.