Microbial forensics is a new discipline combining microbiology and forensic science. Unlike public health investigations, microbial forensics goes further to associate the source of the causative agent with a specific individual or group. Microbial forensics measures molecular variations between related microbial strains and their use to infer the origin, relationship, or transmission route of a particular microbial strain. Several advanced molecular techniques and practices including molecular phylogeny, whole genome sequencing, microarray analysis, and DNA fingerprinting offer reliable results for interpretation in a microbial forensic investigation. Results from such analyses may be related to the intentional use of microbial agents for bioterrorism or the accidental release of any offensive microorganisms or toxins of public health importance specifically for the purpose of determining the origin. Thus, the new discipline of microbial forensics is an integration of an array of well-established fields, such as microbial genomics, phylogenetics, forensic informatics, and classical microbiology. In this article, we review the concept, technology base, success stories, and challenges of microbial forensics and its application in bioterrorism-related investigations.