Due to the important impact that biotechnology has on current Western societies, well-informed critical citizens are needed. People prepared to make conscious decisions about aspects of biotechnology that relate to their own lives. Teachers play a central role in all education systems. Thus, the biotechnological literacy of preservice teachers is an important consideration as they will become an influential collective as future teachers of the next generation of children. The attitudes toward science (and biotechnology) that teachers have affect their behavior and influence the way they implement their daily practice of science teaching in school. This study analyzes the attitudes and knowledge of Spanish preservice teachers toward biotechnology. We designed a new survey instrument that was completed by 407 university students who were taking official degree programs in preschool and primary education. Our results point out that although they are aware of biotechnology applications, topics concerning the structure of DNA, management of genetic information inside the cell, genetically modified organism technology and the use of microorganisms as biotechnological tools were not correctly answered. According to our attitude analysis, Spanish preservice teachers could be defined as opponents of genetically modified product acquisition, supporters of biotechnology for medical purposes and highly interested in increasing their knowledge about biotechnology and other scientific advances. Our results show a positive correlation between better knowledge and more positive attitudes toward biotechnology. A Spanish preservice teacher with positive attitudes toward biotechnology tends to be a student with a strong biology background who scored good marks in our knowledge test.