The ESKAPE pathogens (, , , , , and ) represent clinically important bacterial species that are responsible for most hospital-acquired drug-resistant infections; hence, the need for rapid identification is of high importance. Previous work has demonstrated the suitability of liquid extraction surface analysis mass spectrometry (LESA MS) for the direct analysis of colonies of two of the ESKAPE pathogens ( and ) growing on agar. Here, we apply LESA MS to the remaining four ESKAPE species ( E745, KP257, AYE, and S11) as well as V583 (a close relative of ) and a clinical isolate of AC02 using an optimized solvent sampling system.