Microbes are everywhere. One teaspoon of soil contains an estimated 100 million to one billion bacteria. There are 100 million times more bacteria in the ocean than stars in the known universe. And microbes associated with the human body outnumber human cells ten to one. Microbes communicate with their environment through small molecules, also referred to as secondary metabolites. These microbial metabolites modulate cell to cell communication, which affects biological processes such as cellular differentiation within a colony, virulence, and the homeostatic balance between host health and disease. Microbes are everywhere. One teaspoon of soil contains an estimated 100 million to one billion bacteria. There are 100 million times more bacteria in the ocean than stars in the known universe. And microbes associated with the human body outnumber human cells ten to one. Microbes communicate with their environment through small molecules, also referred to as secondary metabolites. These microbial metabolites modulate cell to cell communication, which affects biological processes such as cellular differentiation within a colony, virulence, and the homeostatic balance between host health and disease. The thesis begins with the introduction of matrix assisted laser desorption ionization-time of flight imaging mass spectrometry (MALDI-TOF IMS). This mass spectrometry based tool is used to visualize the two dimensional distribution of metabolites associated with microbial colonies. Chapter 2 presents the profiling of microbial metabolites by MALDI -TOF MS and the application of IMS to further understand the regulation and production of secondary metabolites in Bacillus subtilis, and the discussion of the biological implications. Chapter 3 introduces mass spectrometry based molecular networking as a strategy to quickly identify the "known unknowns" within complex samples. Chapter 4 proposes the application of molecular networking described in the dissertation to investigate honey bee colony collapse disorder