Polycyclic aromatic hydrocarbons (PAH) have been reported as acetylcholinesterase (AChE) inibitors, although in vitro studies on PAH effects on AChE activity are scarce and have only been performed using electric eel brain extracts. Thus, this study investigated PAH effects on brain AChE activity in a tropical fish species in Southeastern Brazil, mullet (Mugil liza). Mullet specimens were obtained from Guanabara Bay (N = 20), Rio de Janeiro, Brazil. Brain AChE was extracted and exposed to an environmentally relevant concentration of Pyrene, Chrysene, Phenanthrene, and Naphthalene, and PAH metabolites, 2-Naphthol and 1-OH-Pyrene. AChE activity inhibition was observed, although no difference was observed between high- and low- molecular weight PAH. 2-Naphthol was a less potent AChE inhibitor than Naphthalene, albeit non-significantly. Further studies are required, since only one PAH concentration was used herein. Mullet brain extracts seem to be adequate to assess possible neurotoxic PAH effects on fish AChE.