National education reform highlights evolution as a foundational concept of biological literacy. However, introduction of evolutionary theory and practice into microbiology classrooms lags behind use in general biology courses. We used a theme of antibiotic resistance and evolutionary applications in an upper-level undergraduate microbial genetics laboratory course with the goals of (1) increasing students’ ability to explain the details of microbial evolution and (2) increasing students’ acceptance and value of microbial evolution. We presented core concepts and applications of microbial evolution during the course’s weekly lecture. One half of the class also completed a three-week E. coli evolution experiment (“treatment” group) while the other half completed only the traditional exercises (“control” group). We assessed the effectiveness of these activities using pre and post attitude surveys and open-ended content questions about mutation timing and the evolutionary roles of variation, inheritance, and selection. Students in both sections highly accepted and valued evolution at the beginning of the semester and had slight, but nonsignificant, positive attitude gains at the end of the semester. Surprisingly, scores on the content questions did not improve by the end of the semester for either the control or the treatment groups. We attribute this in part to a lack of grade-based incentives and students’ seemingly less earnest attempts on the post assessment. Of greater interest to the development of microbial evolution curricula and assessment, we also observed the potential for traditional microbiological instruction to inadvertently reinforce misconceptions and confound definitions of the key terms “variation” and “selection.” To develop suggestions for teachers to avoid these issues, we are continuing to investigate subdiscipline-specific definitions and language use. This study highlights the continued need for microbial evolution teaching and assessment tools.