<p>Bulk proteomics has been demonstrated to differentiate subpopulations based on molecular phenotypes within bacterial colonies, yet advanced analyses by mass spectrometry imaging (MSI) hold even greater promise for the future. This technology can enable high-throughput spatial phenotyping that can directly visualize distinct components of various biomolecular mechanisms with high mass-resolving power in high spatial resolution analyses. Here, we applied MSI for intact protein imaging directly from thin cross-sections of a biofilm of <i>Bacillus subtilis</i> and after minimal preparation we detected more than 285 unique isotopic envelopes corresponding to unique proteoforms. We paired our MSI analyses with bulk top-down proteomics (TDP) to form extensive experimental libraries, which provided us with high confidence MSI annotations based upon isotopic matching to validated post-translational modifications (PTMs) and truncations. This joint application of MSI and TDP allowed us to describe the microscale spatial proteomic landscape within the <i>B. subtilis</i> biofilm<i>.</i> This study further demonstrated the feasibility of detecting differentiated subpopulations of cells through the identification of proteoforms of cannibalistic protein toxins as well as those involved in active sporulation to highly localized areas within the central and outermost periphery of the biofilm.</p> Graphical abstract <p></p>