Research in fluorescence microscopy presents new challenges, especially with respect to the development of new metal-based fluorophores. In this work, the news fac-[Re(CO) 3 (bpy)L]PF 6 (C3) and fac-[Re(CO) 3 (dmb)L]PF 6 (C4) complexes, where L is an ancillary ligand E-2-((3-amino-pyridin-4-ylimino)-methyl)-4,6-diterbutylphenol, both exhibiting an intramolecular hydrogen bond, have been synthesized for its use as preliminary probes for fluorescence microscopy. The complexes were characterized using chemical techniques such as UV-Vis, 1 H-NMR, TOCSY, FT-IR, cyclic voltammetry, mass spectra (EI-MS 752.22 M + for C3 and 780.26 M + for C4) and DFT calculations including spin-orbit effects. The electron withdrawing nature of the ancillary ligand L in C3 and C4 explains their electrochemical behavior, which shows the oxidation of Re I at 1.84 V for C3 and at 1.88 V for C4. The UV-vis absorption and emission properties have been studied at room temperature in acetonitrile solution. The complexes show luminescent emission with a large Stokes shift (λ ex = 366 nm; λ em = 610 nm for C3 and λ ex = 361 nm; λ em = 560 nm for C4). The TDDFT calculations suggest that an experimental mixed absorption band at 360 nm could be assigned to MLCT (d(Re) →π*(dmb))and LLCT (π(L)→π*(dmb)) transitions. We also assessed the cytotoxicity of C3 and C4 in an epithelial cell line (T84). We found that 12.5 µg/ml of C3 or C4 is the minimum concentration needed to kill the 80% of cell population, as determined by neutral red uptake. Finally, the potential of C3 and C4 as biological dyes for use in fluorescent microscopy was assessed in bacteria (Salmonella enterica) and yeasts (Candida albicans and Cryptococcus spp.), and in an ovarian cancer cell line (SKOV-3). We found that, in all cases, both C3 and C4 are suitable compounds to be used as fluorescent dyes for biological purposes. In addition, we present evidence suggesting that these rhenium (I) tricarbonyl complexes may be also useful as differential fluorescent dyes in yeasts (Candida albicans and Cryptococcus spp.), without the need of antibodies.