Diagnosis of infectious diseases in primary care is predominantly based on medical history and physical examination, as conventional laboratory investigations are often associated with delays that are unacceptable in medical practice. Point-of-care testing, and especially lab-on-a-chip (LoC) systems, are expected to result in a considerable reduction in associated healthcare costs and lead to fast, but appropriate and effective, personalized therapy. Although appropriate sample preparation is essential for final detection, most microfluidic-based approaches start from samples prepared by conventional laboratory procedures, therefore continuing to restrict the use of these systems to a laboratory setting. The lack of integrated sample preparation, especially for sample volumes in the milliliter range, is a major drawback of existing LoC systems. LoC systems that start with real samples and perform a full protocol from sample to result are still rare. In this article, the most recent advances in on-chip sample preparation are reviewed for microfluidic-based diagnosis of infectious diseases.