Mosquitoes transmit serious diseases, which threaten humans and severely affect livestock. The half-lethal concentration (LC₅₀) was calculated by log probit analysis. The LC₅₀ and LC₉₀ values of larvicidal activity of Cassia fistula Linn. hexane-methanol soluble fraction (HMSF) after 24 h of exposure were 21.04 and 34.68 µg/ ml, respectively. The LC₅₀ values after 24 h of exposure were 84.09 µg/ml and 108.08 µg/ml for chloroform–methanol soluble fraction (CMSF) and ethyl acetate-methanol soluble fraction (EMSF) respectively. The percent hatchability of eggs exposed to the hexane extract was 90 ± 5.0, 68.33 ± 7.6, 46.6 ± 11.5, 10 ± 0.0, and 0 ± 0.0% at 10, 20, 40, 60, and 80 ppm, respectively. The pupicidal activity of the hexane extract at 40 µg/ml was 0.0%. The LC₅₀ value of adulticidal activity of the hexane extract was 12.8 mg/test tube. The biosafety of the hexane extract was assessed in nontarget organisms, i.e., zebrafish (Danio rerio) embryos and normal lung cells (BEAS-2B). The hexane extract of C. fistula was well tolerated by zebrafish embryos, and no mortality or toxicity was found in the embryos exposed to the highest tested concentration of 300 µg/ml. Similarly, all the concentrations tested against the normal lung cells (BEAS-2B) showed more than 95% survival. The gas chromatography–mass spectroscopy analysis identified 12 compounds, and 2-methyl hexanoic acid and 2-methyl butanoic acid were the major compounds identified in the hexane extract. The larvicidal activity of C. fistula extracts will help in the development of natural substitutes for vector management of mosquito populations.