Background: Pulegone (PUL) is one of the major constituents of peppermint and pennyroyal. Objective: The main purpose of this study was to investigate the early effects of toxicity of PUL. Materials and Methods: We assessed single doses of toxicity of PUL (2, 0.3 and 0.05 g/kg body weight), administrated by gavage in C57BL/6 mice, evaluated at 1, 6, and 24 h, their clinical status and behavioral, by functional observational battery (FOB), ambulatory conditions, sperm motility, pathological signs, and organ/body weight (O/Bw). Results: No mortality was registered in this in vivo study of oral acute toxicity, in which histological changes were found in selected organs, and PUL mainly showed that the highest concentration reduced mice locomotor activity with significant differences when compared with data of the FOB. Sperm motility also diminished, and hepatic as well as renal alterations were found without modifications in clinical status and O/Bw. Conclusions: We concluded that PUL could be responsible for these findings and consider that FOB is a useful tool to detect early signs of modifications of physiological and biological parameters in mice. Abbreviations Used: PUL: Pulegone, FOB: Functional observational battery, CNS: Central nervous system, OECD: Organization for Economic Cooperation and Development, ANOVA: Analysis of variance, MNI: Mononuclear cell infiltrate, LD50: Median lethal dose