Agricultural crop residues can be converted through thermochemical pyrolysis to bio-oil, a sustainable source of biofuel and biochemicals. The pyrolysis bio-oil is known to contain many chemicals, some of which have insecticidal activity and can be a potential source of value-added pest control products. Brassicacae crops, cabbage, broccoli, and mustards, contain glucosinolates and isocyanates, compounds with recognized anti-herbivore activity. In Canada, canola Brassica napus straw is available from over 6 000 000 ha and mustard Brassica carinata and Brassica juncea straw is available from 200 000 ha. The straw can be converted by microbial lignocellulosic enzymes as a substrate for bioethanol production but can also be converted to bio-oil by thermochemical means. Straw from all three species was pyrolyzed, and the insecticidal components in the bio-oil were isolated by bioassay-guided solvent fractionation. Of particular interest were the mustard straw bio-oil aqueous fractions with insecticidal and feeding repellent activity to Colorado potato beetle larvae. Aqueous fractions further analyzed for active compounds were found not to contain many of the undesirable phenol compounds, which were previously found in other bio-oils seen in the dichloromethane (DCM) and ethyl acetate (EA) solvent phases of the present study. Identified within the most polar fractions were hexadecanoic and octadecanoic fatty acids, indicating that separation of these compounds during bio-oil production may provide a source of effective insecticidal compounds.