Not only do surface-growing microbes such as biofilms display specific traits compared to planktonic cells, but also they display many heterogeneous behaviors over many spatial and temporal contexts. While the application of molecular genetics tools to extract or visualize gene expression or regulatory function data is now common in studying surface growth, the use of analytical chemistry tools to visualize the spatiotemporal distribution of chemical products synthesized by these surface microbes is less common. Here, we review chemical imaging tools that have been used to inform our understanding of surface-growing microbes. We highlight the use of confocal Raman Microscopy, surface-enhanced Raman spectroscopy, matrix-assisted laser desorption/ionization, secondary ion mass spectrometry, desorption electrospray ionization, and electrochemical imaging that have been applied to assess two-dimensional chemical profiles of bacteria. We specifically discuss the use of these tools to study rhamnolipids, alkylquinolones, and phenazines of the bacterium