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Lipids are major functional components of bacterial cells that play fundamental roles in bacterial metabolism and the barrier function between cells and the environment. In an effort to investigate the bacterial lipidome, we adopted a protocol using MALDI-TOF MS imaging coupled to HPTLC to screen a large number of phospholipid classes in a short span of time. With this method, phospholipids of airborne Pseudomonas fluorescens MFAF76a were visualized and identified in sample extracts (measurement accuracy below 0.1 Da, phospholipid identification by means of four characteristic fragment peaks). Via this technique, the P. fluorescens lipidome was shown to comprise three major lipid classes: phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. The protocol described herein is simple, rapid and effective for screening of bacterial phospholipid classes. The remarkable presence of a eukaryotic phospholipid, phosphatidylcholine, was observed in P. fluorescens MFAF76a. This lipid is known to play a role in bacteria–host interactions and had not been known to be found in P. fluorescens cells. [ABSTRACT FROM AUTHOR]