Rhamnolipids, biosurfactants synthesized from natural resources, demonstrate significant applications, including notable insecticidal efficacy against Aedes aegypti L., the primary vector for numerous arboviruses. The global spread of A. aegypti poses substantial public health challenges, requiring innovative and sustainable control strategies. This research investigates the use of andiroba (Carapa guianensis Aubl.) biomass waste as a substrate for synthesizing a rhamnolipid biosurfactant (BSAW) produced by Pseudomonas aeruginosa and evaluates its insecticidal activity against A. aegypti. The findings indicate a biosurfactant yield of 4.42 mg mL⁻¹, alongside an emulsification index approaching 60%. BSAW successfully reduced both surface and interfacial tensions to below 30 mN/m and 4 mN/m, respectively. Characterization revealed that BSAW is a di-rhamnolipid, consisting of two rhamnose units covalently linked to a saturated C₁₀ fatty acid chain. At a concentration of 1.0 mg mL⁻¹, BSAW exhibited notable larvicidal activity, leading to structural impairments and cellular dysfunctions in A. aegypti larvae while also disrupting their associated bacterial microbiota. Moreover, BSAW effectively deterred oviposition in adult mosquitoes. These findings underscore BSAW’s potential to compromise various developmental stages of A. aegypti, supporting integrated arbovirus management approaches. Furthermore, this research emphasizes the feasibility of utilizing agro-industrial waste as substrates for microbial rhamnolipid production.