Gliotoxin is a secondary metabolite of various fungi belonging to the class of epipolythiodioxopiperazines. The mechanism of gliotoxin cytotoxic activity on eukaryotes is established, while the precise interaction between gliotoxin and bacteria has not been clarified yet. The aim of this study was evaluating the gliotoxin action on model Gram-positive and Gram-negative bacteria. It was found that gliotoxin uptake rate was higher in S. epidermidis than E.coli. However, for Gram-negative bacteria, the bactericidal effect is achieved at higher doses of gliotoxin (10-100 μg/ml), compared to Gram-positive ones (0.75-6.25 μg/ml). Bactericidal effect had developed in 4 hours, which was the same for both E. coli MG1655 and S. epidermidis. Novel chip-based bioluminescent sensor with gel-immobilized E.coli MG1655 pKatG-lux revealed oxidative stress. However, pre-incubation of bacteria with Trolox did not affect the bactericidal properties of gliotoxin, while 2-merkaptoethanol and reduced glutathione significantly reduced gliotoxin’s bactericidal property. Another bioluminescent sensor E.coli MG1655 pIbpA-lux revealed heat shock stress in bacteria treated with gliotoxin. At the cellular level, exposure bacteria with gliotoxin accompanied by disruption of bacterial membranes.