Background: The goal of the study is to examine the general public's understanding and perceptions of bioenergy and biofuels in North Carolina (NC) and Tennessee (TN). The study focuses on the public concerns, support and risk evaluations of alternative bioenergy feedstocks and biofuels, and includes an assessment of the economic, environmental, social, and policy impacts of bioenergy production and use. Methods: A sample of consumers in NC and TN were surveyed in the fall of 2013 and spring of 2014 for their perceptions about bioenergy and specifically, biofuels for transportation. Five hundred eighty-six consumers completed the questionnaire electronically (376 in NC and 210 in TN). Results: Respondents reported that the price and vehicle compatibility with biomass-based transportation fuels were the most important factors in their choice of biofuels over gasoline at a pump. Results show that the acceptance of bioenergy depends on the extent of knowledge and available information to consumers about the energy source. A principal component analysis (PCA) indicated seven distinct dimensions of consumer's perception about bioenergy. The key dimensions are the following: how bioenergy benefits the society, risks of bioenergy use, government support for bioenergy, increase in food cost, conditional use of trees, support for low-cost biofuel alternative to current energy, and market attributes of bioenergy purchase. Conclusions: The findings from this study reflect the need for communicating the benefits and risks from the use of bioenergy to the general public through trustworthy channels of communication and targeted policy, market, and institutional support.