In this study, hexasubstituted thiourea was carried out via reaction of isothiocyanato cyclophosphazene intermediates with a series of aromatics amines and amino acids in a one-pot reaction system. The reaction was not as straightforward as typical thiourea synthesis. Six unexpected thiourea derivatives a-f were formed in the presence of cyclotriphosphazene moieties in good yields (53-82%). The structures of a-f were characterized by elemental analysis and FTIR, 1 H, 13 C, and 31 P NMR spectroscopies. The occurrence of reverse thioureas formation in a one-pot reaction system is discussed. The possible binding interaction of the synthesised thiourea a-b in comparison to the predicted phenyl thiourea a-b and the targeted a with enzyme enoyl ACP reductase (FabI) is also discussed. Molecular docking of the targeted hexasubstituted thiourea a is able to give higher binding affinity of −7.5 kcal/mol compared to a-b (−5.9 kcal/mol and −6.3 kcal/mol) and thiourea a-b (−4.5 kcal/mol and −4.7 Kcal/mol).