The Synthesized novel compound of (Z)-2-(5-((10-hexyl-10H-phenothiazin-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl) acetic acid (PTZTZ) was charecterized by nuclear magnetic resonance (1H, 13C), FTIR, UV–Visible, (Total Ion Current) Chromatogram, and ESI full mass spectrum analyses. The determination of DFT analyses of the PTZTZ optimized structure, geometric parameters, UV–Visible Spectroscopy, and FTIR, and their experimental measurement was compared with the resultant estimated results. The frontier molecular orbitals (FMOs), Mulliken's population analysis, and molecular electrostatic potential (MEP) of the title molecule were revealed by theoretical calculations using Gaussian 09 and the fundamental set B3LYP/6-311++G (d,p) level. According to Pharmacological Screening, the title compound showed strong bactericidal activity with a maximum zone of inhibition of (19.8 ± 0.5 mm) at 2.5 g/mL against Proteus mirabilis (MTCC 1771) and scavenging activity analysis of DPPH scavenging trait ranging from (38.8 ± 0.6% to 88.6 ± 4.0%), (H2O2) Hydrogen peroxide exhibited maximal scavenging property of 74.10.2% (p < 0.05), ABTS reducing potential exhibited maximum trait ranging from (39.1 ± 1.2% to 77.8 ± 6.4%), and Ferric reducing antioxidant power (FRAP) exhibited maximum Ferric reducing antioxidant power (FRAP) potential trait ranging from (16.0 ± 0.6% to 43.6 ± 0.5%). Using Discovery Studio 4.0 and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) profile analysis tools, in-silico molecular docking studies revealed the active pocket of the 1HD2 protein to evaluate the evaluation of genotoxicology. Using molecular docking, the compound PTZTZ displayed a greater binding interaction energy of -240.3 (Kcal/mol) against the target protein. The greatest results of this research show that phenothiazine derivatives have superior activity and extremely low binding energy toward the active pocket of protein. In DFT investigations, the title compound demonstrated a minimal energy band gap.