Salmonella and Vibrio cholerae are the deadliest foodborne pathogens, requiring effective routine screening for food safety assurance. Hence, a duplex polymerase chain reaction integrated with a lateral flow immunoassay (PCR-LFIA) was developed for rapid detections of Salmonella and V. cholerae to overcome the laborious, time-consuming, complicated, and costly limitations of standard bacterial culture. The duplex PCR-LFIA could simultaneously detect Salmonella invA and V. cholerae lolB targets at lower limits of 0.1 and 1 ng, respectively. Without requiring culture enrichment, the duplex PCR-LFIA method enabled rapid and simple naked-eye identification of Salmonella and V. cholerae in spiked raw oysters and chicken meat at a limit of detection (LOD) of 106 CFU/mL. A LOD of 104 CFU/mL was achieved by visualizing the PCR amplicons by agarose gel electrophoresis (AGE). This novel duplex PCR-LFIA offers rapid and simple visual detections of pathogenic Salmonella and V. cholerae contaminations, potentially enhancing the capability to monitor and control foodborne illnesses, thereby ensuring food safety.