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Summary: Bacillus spp. produce a wide range of secondary metabolites, including antibiotics, which have been well studied for their antibacterial properties but less so as signaling molecules. Previous results indicated that the lipopeptide bacillomycin D is a signal that promotes biofilm development of Bacillus velezensis SQR9. However, the mechanism behind this signaling is still unknown. Here, we show that bacillomycin D promotes biofilm development by promoting the acquisition of iron. Bacillomycin D promotes the transcription of the iron ABC transporter FeuABC by binding to its transcription factor, Btr. These actions increase intracellular iron concentration and activate the KinB-Spo0A-SinI-SinR-dependent synthesis of biofilm matrix components. We demonstrate that this strategy is beneficial for biofilm development and competition with the Pseudomonas fluorescens PF-5. Our results unravel an antibiotic-dependent signaling mechanism that links iron acquisition to biofilm development and ecological competition. : Natural lipopeptides are known antibiotics produced by Bacillus spp. Xu et al. demonstrate that the lipopeptide antibiotic bacillomycin D is involved in biofilm formation by promoting the acquisition of iron. Versatile functions of bacillomycin D in B. velezensis contribute to the microbe’s ability to compete in its ecosystem. Keywords: Bacillus, bacillomycin D, iron uptake, FeuABC, transcription factor Btr, biofilm, interspecific competition