In the present study, we fabricated a foldable all-in-one microdevice for the nucleic acid test (NAT) performing DNA extraction, polymerase chain reaction (PCR), and on-site colorimetric detection for point‐of‐care (POC) molecular diagnosis of multiple pathogens. The microdevice is composed of three layers. The top layer contains six filter paper discs pre-stored with PCR reagent. The middle layer contains six through-holes corresponding to the positions of six paper discs on the top layer, and was sealed by a flat bottom layer, forming six main chambers where commercially available FTA cards are stored. The intact bacteria samples were introduced into six main chambers for sample treatment – specifically, the isolation, purification, and storage of nucleic acids inside the FTA cards. After the main chambers were filled with water, the top layer was folded towards the middle layer. After 30 thermal cycles for PCR, targets were colorimetrically detected on chip by loading silver nitrate solution into the main chambers and illuminated by UV at 234 nm for 30 s. To examine the versatile applicability, the microdevice was used for the direct detection of three major foodborne pathogens: Salmonella spp., Staphylococcus aureus, and Escherichia coli O157:H7. The feasibility of the microdevice was also tested for detecting multidrug-resistant bacteria, Acinetobacter baumannii. Using the microdevice, multiple pathogens were detected with high specificity and sensitivity of 3.0×102 and 3.0×103 CFU/sample for Gram-negative (Salmonella spp. and Escherichia coli O157:H7) and Gram-positive (Staphylococcus aureus) bacteria, respectively, in less than 2 h, making the microdevice highly suitable in low-resource settings.