This study assesses the effects of sequential applications of the insecticides Bassa 50EC (fenobucarb-F) and Vitashield 40EC (chlorpyrifos ethyl-CPF), sprayed at concentrations used by rice farmers in the Mekong Delta, on the brain acetylcholinesterase (AChE) in climbing perch fingerlings. After spraying the pesticides on the rice fields, the water concentrations of both insecticides decreased below the detection levels within 3 days. The sequential applications caused significant inhibition on the brain AChE activity in the exposed fish. The inhibition by F was quicker, but less prolonged, than for CPF. The inhibition levels caused by the sequential applications were lower than those caused by only CPF and by a mixture of CPF and F. The results indicate that sequential applications of pesticides could have a negative impact on aquatic organisms and fish yields, with implication for the aquatic biodiversity, local people’s livelihood and the aquaculture industry in the Mekong Delta.