Nosocomial infections are ranked as the second-largest infections. E. coli is the primary causative agent of this dreadful infection. To develop effective antibiotic substances against hospitalacquired infections, herein a novel carbon monoxide tagged Ru(II) complex of N-o-hydroxyacetophenone-ethylenediamine-N -o-vanillin is reported as an effective antimicrobial agent of its form. In addition to molecular structure elucidation, an explanation has been made about the related charge topography concerning the biological effect. The formulation of the complex based on hyphenated experimentaltheoretical evaluation suggests an octahedral structure. Density functional study was carried out confined to the formalism as B3LYP functional/LanL2DZ basis set for Ru atom and 6-31 g(d,p) for the rest of the atoms. The stable form of geometry and other electron density plots are the prominent inclusions discussed in this work. The primary characterization techniques that have been used for the formulation include elemental analysis, mass spectrometry, nuclear magnetic resonance (C and H), UV-Vis, and FT-IR. The experimentally observed data was validated with theoretical/computational outputs. Both the experimental and theoretical results sound in excellent agreement when compared at each step of the characterization. A suitable trans-octahedral geometry has been suggested for the complex. From the antimicrobial actions against nosocomial E. coli, an MIC value of 30 μM was obtained for the selected compound and hence can be assumed as a standard novel antibiotic substance against nosocomial infections. The respective microbial study has been discussed, keeping in view the microbial growth study, the qualitative and quantitative analysis to add fascination to the work being reported.