Biology I is a required course for many science, technology, engineering, and mathematics (STEM) majors and is often their first college-level laboratory experience. The replacement of the traditional face-to-face laboratory experience with virtual laboratories could influence students' content knowledge, motivation to learn biology, and overall course performance. Three hundred undergraduate STEM students in face-to-face or virtual laboratories in a Biology I course completed a laboratory content test and the Biology Motivation Questionnaire II at the beginning and end of the semester. Final course grades were also obtained. Analyses revealed no significant differences between STEM students in the face-to-face and virtual laboratories in learning gains on the content test and final course grades. Two thirds of the STEM students experienced a decline in motivation to learn biology over the semester, but no significant differences were found between the laboratory groups. Thus, virtual laboratories may offer an affordable alternative to resource intensive face-to-face laboratories in large-enrollment Biology I courses.