Top-down identification of proteins by liquid extraction surface analysis (LESA) mass spectrometry has previously been reported for tissue sections and dried blood spot samples. Here, we present a modified "contact" LESA method for top-down analysis of proteins directly from living bacterial colonies grown in Petri dishes, without any sample pretreatment. It was possible to identify a number of proteins by use of collision-induced dissociation tandem mass spectrometry followed by searches of the data against an E. coli protein database. The proteins identified suggest that the method may provide insight into the bacterial response to environmental conditions. Moreover, the results show that the "contact" LESA approach results in a smaller sampling area than typical LESA, which may have implications for spatial profiling.