Honey is a natural pure food produced by honeybees from the nectar of various plants, and its chemical composition includes carbohydrates, water, and some minor compounds, which are very important for honey quality and authentication. Most of honey’s minor components are related to the botanic origin, climate, and geographic diversity. In this work, we report an original case study on monofloral honey samples of twelve different botanic origins produced in Tuscany (Italy) based on the ‘semi-quantitative’ analysis of emission, excitation, and synchronous front-face fluorescence spectra. This is the first front-face fluorescence study of Italian honey samples and, to our knowledge, the first fluorescence investigation of honey from inula (Inula viscosa (L.) Aiton), marruca (Paliurus spina-christi Mill.), lavender (Lavandula L. 1753), sulla (Hedysarum coronarium L.), arbutus (or strawberry tree) (Arbutus unedo L., 1753), and alfalfa (Medicago sativa L.) plants. Results obtained from fluorescence spectroscopy are discussed in terms of characteristic spectral emission profiles typical of honey of different botanic origins. Moreover, the spectral analysis based on the decomposition of the front-face fluorescence (FFF) spectra in terms of single main fluorophores’ components is here proposed to identify several minor compounds, such as amino acids, phenolic acids, vitamins, and other fluorescent bioactive molecules.