<p>The widespread use of synthetic insecticides has led to environmental concerns and the development of resistance in mosquito populations, prompting the need for alternative control strategies. This study explores the mosquito larvicidal efficacy of a β-isocostic acid-based formulation against <i>Aedes aegypti</i> and <i>Culex quinquefasciatus</i>. β-isocostic acid, isolated from the leaves of <i>Sphaeranthus indicus</i> using hexane extraction, was prepared in a mineral oil formulation and tested in laboratory conditions at varying concentrations (0.5, 1.0, 1.5, and 2.0 ppm). The study evaluated lethal concentrations (LC₅₀ and LC₉₀) and the effects of the formulation on non-target organisms, <i>Diplonychus indicus</i> and <i>Gambusia affinis</i>. Results demonstrated significant larvicidal activity, with LC₅₀ values of 0.90 ppm for <i>Ae. aegypti</i> and 0.81 ppm for <i>Cx. quinquefasciatus</i>. For comparison, an azadirachtin-based formulation showed LC₅₀ values of 0.46 ppm and 0.33 ppm, respectively. Additionally, the β-isocostic acid formulation had no adverse impact on non-target organisms at 1 ppm, suggesting it could be integrated into mosquito control programs without harming natural predators. These findings indicate that β-isocostic acid-based formulations have substantial potential for eco-friendly mosquito control, especially for larvae of vector species. The study underscores the importance of botanical insecticides as sustainable alternatives to synthetic pesticides in Integrated Mosquito Management strategies.</p>