The lipopeptoid-peptide chimera (LPP) substance library, synthesized by consecutive Ugi four-component reactions (U-4CR), is comprised of derivatives from formic acid (C1) to arachidic acid (C20). A further derivatization of the LPPs with a fluorescent dye a/o guanididium moieties and the dimerization of larger LPP fragments with a small diisocyanide was additionally accomplished. A facile 24 h-luminescence assay, based on gram-negative luminescent bacteria Aliivibrio fischeri, was developed. In combination with the new 2D-heatmap plot, a quick distinction between acute toxic and chronic antibacterial compounds can be made. Several LPPs showed promising antimicrobial activity with IC50 values in the range of 1 μM and 10 μM in this and other bioassays against different microorganisms like Bacillus subtilis, Septoria tritici, Botrytis cinerea and Phytophtora infestans.