Greenhouse gas emissions are a major contributor to climate change, which originate from several sectors, including energy, transportation, manufacturing, and construction. However, the transportation sector accounts for approximately 25% of total emissions, indicating the urgent need for effective mitigation strategies. One potential solution is the use of renewable energy, particularly biofuels, which can reduce carbon dioxide emissions by partially replacing fossil fuels. In Indonesia, this transition is supported by Government Regulation No. 79/2014 on the National Energy Policy, including the mandatory B20 program that promotes biodiesel blending. Therefore, this study aims to examine public knowledge, attitudes, and perceptions regarding biofuel use in the transportation sector in Indonesia. Data were collected through a questionnaire survey involving 256 respondents and analyzed using analysis of variance (ANOVA) to assess differences across demographic factors. The results showed that respondents under 18 years of age had knowledge levels comparable to those aged 18–24, while significant differences were observed among older age groups. Educational background influenced knowledge, with vocational high school and university graduates accounting for 28% and 40%, respectively. Gender differences were also identified, as male respondents showed slightly higher knowledge levels (54%) than females (46%). Despite showing positive attitudes toward biofuels, public knowledge remained limited, with more than 60% of respondents unaware of natural resource and energy management issues. Public attitudes and perceptions were predominantly positive, with more than half of respondents expressing agreement or strong agreement. These results showed the need for strengthened public education to support effective biofuel policy implementation.