Rapid and sensitive detection of pathogenic bacteria in various samples, including food and drinking water, is important to prevent bacterial diseases. Most bacterial solutions contain only a small number of bacteria in complex matrices with impurities; hence, pretreatment is necessary to separate and concentrate target bacteria before sensing. Among various pretreatment methods, iron oxide magnetic nanoparticle (MNP)-based pretreatment has drawn attention owing to the unique properties of MNP, such as high magnetic susceptibility, superparamagnetism, and biocompatibility. After target bacteria are captured by recognition molecule-functionalized MNPs, bacteria–MNP complexes can be easily separated and enriched by applying an external magnetic field. Various devices, such as optical, electrochemical, and magnetoresistance sensors, can be used to detect target bacteria, and their detection principles have been discussed in numerous review papers. Herein, we focus on recent research advances and challenges in magnetic pretreatment of pathogenic bacteria using microfluidic devices, which offer the advantages of process automation and miniaturization.