Infections caused by antibiotic-resistantStreptococcus pneumoniaeare of growing concern for healthcare systems, which need new treatment options. Screening microorganisms in terrestrial environments has proved successful for discovering antibiotics, while production of antimicrobials by marine microorganisms remains underexplored. Here we have screened microorganisms sampled from the Oslo Fjord in Norway for production of molecules that prevent the human pathogenS. pneumoniaefrom growing. A bacterium belonging to the genusLysinibacilluswas identified. We show that this bacterium produces a molecule that kills a wide range of streptococcal species. Genome mining in BAGEL4 and AntiSmash suggested that it was a new antimicrobial compound, and we therefore named it lysinicin OF. The compound was resistant to heat (100 °C) and polymyxin acylase but susceptible to proteinase K, showing that it is of proteinaceous nature, but most probably not a lipopeptide.S. pneumoniaebecame resistant to lysinicin OF by obtaining suppressor mutations in theamilocus, which encodes the AmiACDEF oligo peptide transporter. We created ΔamiCand ΔamiEFmutants to show that pneumococci expressing a compromised Ami system were resistant to lysinicin OF. Furthermore, by creating mutants expressing an intact but inactive Ami system (AmiED184A and AmiFD175A) we could conclude that the lysinicin OF activity depended on the active form (ATP-hydrolysing) of the Ami system. Microscopic imaging and fluorescent labelling of DNA showed thatS. pneumoniaetreated with lysinicin OF had an average reduced cell size with condensed DNA nucleoid, while the integrity of the cell membrane remained intact. The characteristics and possible mode of action of lysinicin OF are discussed.