A recent scientific discipline, bioinformatics, defined as using informatics for the study of biological problems, is now a requirement for the study of biological sciences. Bioinformatics has become such a powerful and popular discipline that several academic institutions have created programs in this field, allowing students to become specialized. However, biology students who are not involved in a bioinformatics program also need a solid toolbox of bioinformatics software and skills. Therefore, we have developed a completely online bioinformatics course for non-bioinformaticians, entitled “BIF-1901 Introduction à la bio-informatique et à ses outils (Introduction to bioinformatics and bioinformatics tools),” given by the Department of Biochemistry, Microbiology, and Bioinformatics of Université Laval (Quebec City, Canada). This course requires neither a bioinformatics background nor specific skills in informatics. The underlying main goal was to produce a completely online up-to-date bioinformatics course, including practical exercises, with an intuitive pedagogical framework. The course, BIF-1901, was conceived to cover the three fundamental aspects of bioinformatics: (1) informatics, (2) biological sequence analysis, and (3) structural bioinformatics. This article discusses the content of the modules, the evaluations, the pedagogical framework, and the challenges inherent to a multidisciplinary, fully online course. © 2017 by The International Union of Biochemistry and Molecular Biology, 2017.