Methylisothiazolinone (MIT) is widely used as biocide in hair and skin care products and in high concentrations, more than 150 mg/L, in cooling towers in the paper mill and refinery industry. This product has been recognized as a potent contact allergen and is discharged to the wastewater treatment plants (WWTP) from industrial processes or domestic usages. However, the role of MIT in biological treatment has not been characterized, and therefore, the effect of MIT on the composition and performance of activated sludge has been analyzed. For this purpose, a sequential batch reactor was fed with a synthetic solution containing 5 mg/L of MIT. Addition of MIT had shown no significant effect on organic matter metabolization (dissolved organic carbon [DOC] removal remained constant at 80-90%). On the contrary, a loss of efficiency in the nitrification process occurred (ammonium removal decreased from 90% to 20% per cycle throughout the experiment), first affecting the nitrite oxidizing bacteria and, after around 40 cycles, also the ammonium-oxidizing bacteria disappeared, as shown by fluorescence in situ hybridization analysis. Bioindicative studies based on counts of protozoa and sludge biotic index indicated that, at the end of the process, a stable and well-colonized protozoa community was obtained. Finally, respirometric tests indicated some acclimatization of the heterotrophic bacteria to MIT, as shown by shorter lag periods when the sludge was previously fed with MIT. The role of MIT in biological treatment has been characterized to increase the knowledge about this pollutant effects on activated sludge to improve WWTP performance.