Bacterial pathogens are one of the leading causes of food safety incidents and product recalls worldwide. Timely detection and identification of microbial contamination in agricultural and food products is crucial for disease prevention and outbreak investigation. In efforts to improve and/or replace time-consuming and laborious “gold standards” for pathogen detection, numerous alternative rapid methods have been proposed in the past 15 years, with a trend toward incorporating nanotechnology and nanomaterials in food pathogen detection. This article is a review of the use of nanotechnology in various detection and sample preparation techniques and advancements in nanotechnology applications in food matrices. Some practical considerations in nanobioassay design are discussed, and the gaps between research status quo and market demands are identified.