The antimicrobial agent, triclosan, has been designated as a “contaminant of emerging concern (CEC)”. Previous in vivo studies have shown that triclosan exposure can inhibit acetylcholinesterase (AChE) activity. However, mechanistic insights into AChE inhibition by triclosan are missing. Here, using in vitro activity assay with purified AChE, we show that triclosan can directly inhibit AChE. In vivo, triclosan exposure resulted in reduced total antioxidant capacity concomitant with reduced AChE activity in the adult zebrafish brain. Adult zebrafish when pre-treated with antioxidant melatonin, resulted in attenuated oxidative stress and attenuated inhibitory effect of triclosan on the AChE activity. Our results indicate that triclosan can affect AChE activity both by direct binding and indirectly through increased oxidative stress and therefore, provide important mechanistic insights into triclosan induced neurotoxicity. © 2021 Elsevier B.V.