The synthesis, in silico and in vitro antimicrobial studies of N-(7-nitro-10H-phenothiazin-3-yl) benzenesulphonamide derivatives against some microorganisms were reported. The derivatives were prepared through the reaction of 1-amino-4-chlorobenzene with 1-amino-4-nitrobenzene to form N¹-(4-nitrophenyl) benzene-1, 4-diamine. The treatment of this with sulphur and catalytic amount of iodine generated 7-nitro-10H-phenothiazin-3-amine. This intermediate was further treated with appropriate amount of sulphonyl chlorides to yield the final products, N-(7-nitro-10H-phenothiazin-3-yl) benzenesulphonamide derivatives. The derivatives were analyzed via FTIR, ¹H NMR, ¹³C NMR and HRM spectroscopic techniques and the results were consistent with the assigned structures. In silico antimicrobial studies were done via Autodock software to predict the binding energies of the prepared derivatives with the active sites of the target receptors 7D8I, 7R1M, 7AB4, 4YXB, 5ZVP for S. aureus, B. cereus, E. coli, S. typhi, A. fumigatus. The binding energies for the in silico antimicrobial studies were in the range -8.8 to -5.8 kcal/mol. The zones of inhibition for in vitro antimicrobial activity were in the range 18 to 33.5 mm. The synthesized compound showed promising biological activities.