Detecting foodborne pathogens, particularly bacteria, in complex food samples with the use of biosensors capable of swift, accurate, and efficient detection and quantification is essential to ensuring food safety and public health because it aids in the diagnosis of foodborne illness. Nevertheless, the diagnostic testing of handheld sensing devices for pathogenic bacteria recognition in real-world samples continues to pose a challenge in this domain. Interestingly, using engineered functionalized nanomaterials in conjunction with microfluidic technology is effective in the early detection of foodborne diseases by directly identifying and quantifying pathogenic bacteria present in contaminated food samples. This book chapter provides a comprehensive overview of microfluidic-assisted biosensors, including the assays centered around specially designed functionalized upconversion nanoparticles, functionalized carbon nanomaterials, functionalized quantum dots, functionalized metal nanoparticles, and functionalized magnetic nanoparticles for the pathogenic bacteria detection. Finally, the key obstacles to translating research findings into commercial sensing device development are described.