Comparison studies on the acetalization of 10-methyl-10H-phenothiazine-carbaldehyde with 1,3-propanediol, or 2-substituted-1,3-propanediols, under conventional versus microwave assisted conditions and standard organic solvents versus water, were performed as an attempt toward more environmentally benign synthetic methods. New 3-(1,3-dioxan-2-yl)-10-methyl-10H-phenothiazine derivatives were obtained in high yields by azeotropic distillation of water and in moderate yields by microwave assisted synthesis in different solvents, including water under superheated conditions. The solvent influence upon stabilizing the key intermediates involved in the acetalization mechanism was assumed based on DFT calculations, which indicated a favorable enthalpy profile in water solvent. Structural investigations of the new compounds based on spectroscopic methods (NMR, FT-IR, UV–vis, and MS), were completed with molecular mechanics and semi-empirical DFT calculations, which supported an anancomeric chair conformation of the 1,3-dioxane ring with the phenothiazine substituent in the equatorial position and possible free rotation about the single bond linking the two heterocyclic units. The new compounds display daylight fluorescence characterized by remarkably large Stokes shifts determined by LE spectroscopy.