The present study aimed to evaluate the phytochemical constituents of hydro-alcoholic oak bark extract using gas chromatography–mass spectrometry (GC–MS) analysis and to determine its acute toxicity (LD50) in female local rabbits. Oak bark was extracted using 70% ethanol, yielding 11.7% dark brown crystalloid extract. GC–MS analysis identified several biologically active compounds, with gallic acid representing the predominant constituent (49.06%), followed by linalool (7.74%), isobornyl acetate (6.12%), 3-deoxy-D-mannonic acid (4.02%), salicylic acid (3.76%), and α-bisabolol (3.22%). These compounds are known for their antioxidant, anti-inflammatory, antimicrobial, and tissue-protective activities. The acute toxicity study was conducted using the Up-and-Down method in female local rabbits. The estimated oral LD50 value of the oak bark extract was (1850.7) mg/kg body weight, indicating slight to moderate toxicity according to toxicity classification standards. Clinical signs observed in intoxicated animals were dose-dependent and included mild sedation, decreased locomotor activity, piloerection, hypothermia, bradycardia, salivation, muscular weakness, anorexia, tremors, convulsions, respiratory depression, and death at higher doses. Animals receiving lower doses recovered within 18–25 hours, whereas higher doses caused severe neurotoxic manifestations and mortality. The findings of the current study demonstrate that oak bark hydro-alcoholic extract contains numerous bioactive phytochemicals with potential therapeutic value. However, administration of high doses may induce significant toxic effects, particularly on the nervous and respiratory systems.