Aflatoxin B1 (AFB1) is the most widespread mycotoxin, and it is a feed contaminant and is highly toxic, causing carcinogenic, mutagenic, and teratogenic effects. Many researches clarified the peripheral effects of the exposition to AFB1; however, there are few studies explaining their effects on central nervous system. The aim of the present study was to evaluate the effects caused by acute oral administration of AFB1 on behavioral tests and selected biochemical parameters. Young male Wistar rats received a single administration of AFB1 (250 µg/kg/i.g.) and 48 hours thereafter they were subjected to behavioral analysis. After the tests, biochemical parameters were measured in the cerebral cortex. Acute treatment with AFB1 caused neurotoxic effects, evidenced by a significant reduction in the levels of non-enzymatic antioxidant defenses, ascorbic acid, and non-protein sulfhydryl groups. In addition, AFB1 increased protein kinase C (PKC) activation, evidenced by an increase in phosphorylation of Ser of PKCα. In this acute protocol, a single oral administration of AFB1 was able to cause changes in important neurochemical parameters, without concomitant, detectable behavioral alterations. These results reinforce that monitoring mycotoxin levels in food is essential to guarantee food security.