Matrix‐assisted laser desorption/ionization imaging of biofilms cultured on agar plates is challenging because of problems related to matrix deposition onto agar. We describe a one‐step, spray‐based application of a 2,5‐dihydroxybenzoic acid solution for direct matrix‐assisted laser desorption/ionization imaging of hydrated Bacillus subtilis biofilms on agar. Using both an optimized airbrush and a home‐built automatic sprayer, region‐specific distributions of signaling metabolites and cannibalistic factors were visualized from B. subtilis cells cultivated on biofilm‐promoting medium. The approach provides a homogeneous, relatively dry coating on hydrated samples, improving spot to spot signal repeatability compared with sieved matrix application, and is easily adapted for imaging a range of agar‐based biofilms. Copyright © 2016 John Wiley & Sons, Ltd.