Two alternative extraction media for botanical insecticide nicotine were evaluated in order to increase its recovery efficiency from tobacco leaves: oleic acid oil-in-water (O/W) emulsions and oleic acid. The use of oleic acid O/W emulsions stabilized by calcium caseinate as extraction media in a percolation process showed a recovery efficiency between 62% (25 °C) and 68% (60 °C). Oleic acid O/W emulsions stabilized by sodium dodecyl sulfate (SDS), Tween 20, Tween 80 or Triton X100 were inappropriate as extraction media since they presented flocculation or phase separation after the extraction procedure. Bioassays of nicotine oleate O/W emulsions stabilized by sodium caseinate showed a lethal time 50 (LT50) of 10 min. Nicotine extraction by a modified effleurage method showed a recovery efficiency between 85% (25 °C) and 95% (60 °C). Nicotine oleate O/W emulsions (45 vol %) prepared with the effleurage extract and using SDS showed the highest bioactivity (LT50=20 min). Nicotine oleate O/W emulsions prepared with the effleurage extract and stabilized by sodium caseinate, Tween 20, Tween 80 or Triton X100 showed LT50 values of over 50 min. Interfacial tension measurements of nicotine effleurage extract-aqueous surfactant solution interfaces, droplet size analysis of nicotine oleate emulsions and nicotine concentrations at emulsion aqueous phases suggest that nicotine oleate in O/W emulsions stabilized by SDS was encapsulated in oil droplet emulsions, while in sodium caseinate, Tween 20, Tween 80 or Triton X100 emulsions the nicotine oleate acts as a cosurfactant, decreasing its insecticide activity.