The usability of bioluminescence for imaging of single cells is complicated by its low light levels compared to fluorescence measurements and the requirement of exogenous luciferin supply for most luciferases. Bacterial bioluminescence uses reduced flavin mononucleotide as a luciferin that is abundant in all cells. Therefore, this system is purely genetically encodable by the lux operon, which contains the bacterial luciferase and enzymes for substrate recycling. However, the brightness of bacterial bioluminescence is relatively low in comparison to other luciferases. We have generated an improved lux operon named ilux with an approximately sevenfold increased brightness when expressed in E. coli cells. The ilux operon consists of the luxCDABE genes and an additional FMN reductase, which were optimized for enhanced brightness by mutagenesis. ilux allows for imaging of single E. coli cells over timespans up to several days with enhanced spatiotemporal resolution. Since metabolic energy is required to generate auto-bioluminescence light, ilux can be used to assay cellular viability on the single-cell level, as demonstrated by application of different antibiotics.