Novel fluorescent coumarin-thiophene appended Schiff bases have been synthesized in good yields (76-89%). The effects of substitution on the fluorescent intensity and solubility of the molecules were examined with experimental techniques. The deprotonation properties of the prepared Schiff bases were examined, in DMSO, by titration of each of the ligands with TBAOH. We also studied the reverse protonation of the fluorophores using TFA. The structural and electronic properties of the neutral and the deprotonated forms of the fluorophores were also examined in the framework of DFT calculations. Also, the cytotoxicity of each of the compounds was tested against L929 fibroblasts and human lung cancer (small cell). Furthermore, the antimicrobial activity of each of the compounds was studied against both Gram-negative and Gram-positive bacterial and fungal species. In the final studies, the plasmid DNA interactions with the compounds were investigated with agarose gel electrophoresis. The thermal properties of all the prepared compounds were determined by thermal gravimetric analysis (TGA) and were found to be thermostable up to at least 335 degrees C.