Abstract: The julolidine (conventional name for 1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolone)-based compounds have been reported as ligands that can detect Cu²⁺ by colorimetric and fluorometric methods, and as inhibitors of the amyloid-β protein self-assembly (which is associated the pathogenesis of Alzheimer's disease). Furthermore, the presence of this rigid structure has shown to increase the fluorescence quantum yield comparatively to the respective unrestricted dye. Taking this in account, we decided to explore the influence of the julolidine moiety in the properties of benzophenoxazine derivatives, by evaluating the antimicrobial activity against Saccharomyces cerevisiae PYCC 4072 of newly synthesised fluorescent 2,3,6,7-tetrahydro-1H-benzo[a]quinolizino[1,9-hi]phenoxazin-14(5H)-iminium chlorides, in comparison with their analogues. Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the NMR portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER (European Fund for Regional Development)-COMPETE-QREN-EU for financial support to Research Centres CQ/UM [PEst-C/QUI/UI0686/2013 (FCOMP-01-0124-FEDER-037302)] and CBMA (project PEst-OE/BIA/UI4050/2014). Post-doctoral grant to B. R. Raju (SFRH/BPD/62881/2009) is also acknowledged to FCT, POPH-QREN, and FSE.