Medicinal plants harbor numerous natural bioactive compounds with therapeutic potential, utilized in the synthesis of various drug formulations. While the diverse benefits of herbal formulations have been extensively researched, a toxicity assessment is crucial to establish a wide margin of safety for the therapeutic application of this polyherbal formulation. The recent study aimed to assess the phytochemical profile and potential toxic effects of an aqueous extract derived from a polyherbal formulation containing Petroselinum crispum L., Coriandrum sativum L., and Apium graveolens L. The investigation involved acute and sub-acute toxicity studies conducted in male and female Swiss albino mice, as well as adult rats. High-performance liquid chromatography analysis revealed a rich composition of phytochemical compounds in the extract. In acute toxicity assessments, oral administration of the extract up to 14 g/kg showed no signs of toxicity or fatalities in mice. However, intraperitoneal administration resulted in dose-dependent toxicity, with a calculated LD50 of 11.8 g/kg. Sub-acute toxicity studies in rats over 28 days showed no significant changes in organ weights, hematological, or biochemical parameters, except for a minor decrease in WBC count. Histopathological examination revealed no morphological disturbances in the liver and kidneys, indicating a wide margin of safety for therapeutic use.