Physalia physalis, or Portuguese Man O’ War, is one of the most venomous cnidarians. Despite its known toxicity, detailed studies on its venom components remain limited, specifically concerning their neuroactive effects. This study seeks to explore the effects of total/crude venom (CV), as well as high-molecular-weight (HMW) and low-molecular-weight (LMW) venom fractions, using Drosophila melanogaster as an in vivo model. Oral administration enabled the assessment of fly mortality and locomotor behavior. CV and LMW venom fractions induced similar levels of mortality, while HMW and control groups showed comparably lower rates. The CV-treated group displayed acute locomotor impairment, with an acute toxicity pattern in climbing and moderate symptom variability. In contrast, the HMW-treated group showed minimal impairment, characterized by a predominantly straight trailing climbing pattern and reduced symptom variability. The LMW-treated group exhibited pronounced locomotor dysfunction, displaying a variability of abnormal climbing behaviors and symptoms. Real-time monitoring revealed an initial phase of hyperactivity followed by prolonged paralysis, resulting in reduced locomotor activity and altered circadian rhythm. The results indicate that neurotoxicity resides primarily in LMW components, demonstrating the utility of Drosophila for venom screening and neurodegenerative-related research. This work supports future proteomic and transcriptomic studies identifying novel bioactive compounds with potential therapeutic applications.