Reprocessing failure of endoscopes may result in outbreaks of serious infections in vulnerable patients caused by Gram-negative bacteria. P. oleovorans (PSOL) was detected in 6 automated endoscope washer-disinfectors (AEWDs) in two reprocessing units during routine check and probing for quality control. Ten endoscopes were probed yielding the growth of PSOL. Two different PSOL strains were identified by genotyping. Biofilms and planktonic cells of both PSOL (N = 2) and of Pseudomonas aeruginosa PAO1 as reference were incubated with increased disinfectant concentrations modelling the disinfection process in the AEWD. PSOL in planktonic form was eradicated by GLUT1% (1 g/100 g) at 55 °C. GLUT at a higher concentration of 3 % resulted in the eradication of PSOL biofilms at 25 °C. The persistent growth of PSOL in quality controls indicates inadequate disinfection. Increase of the concentration of GLUT would be an option to eradicate PSOL. However, increasing the concentration of GLUT may lead to corrosion of the sensible instruments and toxic side-effects in patients. Further investigation on disinfectant type and concentration, the reservoir of contamination and defining additional disinfection steps are warranted.