Multiplexed diagnostic testing has the potential to dramatically improve the quality of healthcare. Simultaneous measurement of health indicators and/or disease markers reduces turnaround time and analysis cost and speeds up the decision making process for diagnosis and treatment. At present, however, most diagnostic tests only provide information on a single indicator or marker. Development of efficient diagnostic tests capable of parallel screening of infectious disease markers could significantly advance clinical and diagnostic testing in both developed and developing parts of the world. Here, we report the multiplexed detection of nucleic acids as disease markers within discrete wells of a microfluidic chip using molecular beacons and total internal reflection fluorescence microscopy (TIRFM). Using a 4 × 4 array of 200 pL wells, we screened for the presence of four target single stranded oligonucleotides encoding for conserved regions of the genomes of four common viruses: human immunodeficiency virus-1 (HIV-1), human papillomavirus (HPV), Hepatitis A (Hep A) and Hepatitis B (Hep B). Target oligonucleotides are accurately detected and discriminated against alternative oligonucleotides with different sequences. This combinatorial chip represents a versatile platform for the development of clinical diagnostic tests for simultaneous screening, detection and monitoring of a wide range of biological markers of disease and health using minimal sample size.