Current state-of-the-art methods require multiple procedures, which are expensive, complex, and time-intensive. Additionally, many laboratories lack the necessary infrastructure to perform comprehensive analyses in-house, relying instead on third-party service providers. We have developed MLSTnext technology, a single-tube-PCR, with simple workflow for microbial pathogens, capable of serotyping, subtyping, surveillance, identifying multiple serotypes as well as an integrating data analysis software without the need for bioinformatics expertise. MLSTnext has been applied to a wide range of food safety applications such as Salmonella, Listeria, Legionella, HAV, STEC/E. coli, Campylobacter, Cyclospora and more. All assays have generated high-resolution results, accurately serotyping to the subtype level. For instance, the Salmonella assay distinguished between two S. typhimurium strains by identifying sequence variations in four loci, revealing the same serotype but different strains. The same assay demonstrated the ability to detect the co-presence of multiple serotypes of Salmonella or Listeria (up to nine serotypes), within a single sample. Furthermore, the unique loci profile enabled effective traceability for contamination surveillance and outbreak management. MLSTnext is versatile and the entire workflow takes 10-24 hours (depending on the sequencing platform) without the need for pure colonies/isolates.