Alternative methods of insect management are an important field of study for agriculture. The current study aimed to determine the effect of aqueous extracts from Simarouba sp. (AE-S) on the biology of Plutella xylostella and to determine the toxicity of the extract to the nematode Caenorhabditis elegans (an important in vivo alternative assay system for toxicological study). Lyophilized AE-S was chemically investigated by Ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). We evaluated the effect of the botanical extract on the life cycle of P. xylostella, from larval stage to adult stage, at concentrations of 10%, 5%, 1%, 0.1%, 0.05%, and 0.01% and a control. Subsequently, we analyzed the toxicity of the extract in an in vivo model. AE-S showed high amount of phenolic and flavonoid compounds. Six compounds were identified based on UHPLC-MS/MS analysis, including flavanone, kaempferol, 4,5-dimethoxycanthin-6-one, 11-acetylamarolide, ailanthinone, and glaucarubinone. The median lethal time for P. xylostella was estimated to be 96 h in all concentrations of AE-S, and at 120 h, 100% of the individuals were dead. Larvae exposed to AE-S at concentrations of 0.01, 0.05, and 0.1% showed a reduction in leaf area consumption, underdevelopment, and reductions in movement and pupal biomass. The lowest concentrations of AE-S (0.1%, 0.05%, and 0.01%) did not cause mortality in nematodes. Thus, the aqueous extract of Simarouba sp. could be an effective control tool because it mainly acts in the larval stage, the stage at which the insect causes damage to brassicaceae.