Pyrolysis converts biomass such as agricultural and forestry waste into bio-oil, preserving some chemicals while creating other, new ones. Nicotine, a chemical present in tobacco leaves and a known pesticide, was found to remain intact during pyrolysis. As expected, insecticidal properties were observed for tobacco bio-oil. Pesticide characteristics of tobacco bio-oil have been observed on the Colorado potato beetle (CPB), a pest currently resistant to all major insecticides, as well as a few bacteria and fungi that do not currently respond well to chemical treatment. Unexpectedly, nicotine-free fractions of the bio-oil were also found to be highly lethal to the beetles and successful at inhibiting the growth of select microorganisms. Through GC-MS, it was found that the active, nicotine-free fractions were rich in phenolics, chemicals likely created from lignin during pyrolysis. While bio-oils in general are known to contain phenolic chemicals, such as cresols, to our best knowledge, quantitative analysis has not been performed to determine if these chemicals are solely responsible for the observed pesticide activities. Based on GC-MS results, ten of the most abundant chemicals, eight of which were phenolic chemicals, were identified and examined through bio-assays. A mixture of these chemicals at the concentration levels found in the bio-oil did not account for the bio-oil activity towards the microorganisms. Tobacco bio-oil may have potential as a pesticide, however, further analyses using liquid chromatography is necessary to identify the remaining active chemicals.