The synergic effect and microstructural analysis using Fourier Transform Infrared (FTIR) spectroscopy, Raman spectroscopy, Brunauer-Emmett-Teller (BET), and Scanning Electron Microscopy (SEM) effectively identify functional groups in cement paste containing Ordinary Portland Cement (OPC) and three additives: Nano-Biomass Silica (NBS), Polycarboxylate Ether (PCE), and a bio-admixture (BA) with Lysinibacillus fusiformis. Strength analysis showed that BA concrete achieved the highest strength at 28 days (62.3 MPa), demonstrating the effectiveness of bio-precipitated CaCO<inf>3</inf>. Bio-admixtures, especially bio-CaCO<inf>3</inf>, significantly improved strength across all stages. Raman and FTIR spectroscopy were employed to evaluate the four phases formed during hydration, correlating phase formation with FTIR band shifts across different curing times (7, 14, and 28 days). The spectra's Oâ��H, Siâ��O, and Câ��O/Câ��H stretching and bending regions identified the samples' constituents and band vibrations. Transmission and attenuated total reflectance methods provided non-destructive testing of OPC, CNBS, CPCE, and CBA samples. Key spectral bands indicated physiological activities such as silicate organization (C-A-S-H or Câ��Sâ��H), sulfate, hydroxylation, nano-SiO<inf>2</inf>, bio-CaCO<inf>3</inf>, water molecules, and carbonation. BET analysis showed CBA with a higher surface area (15.82 m²/g) and lower pore volume (0.0336 cm³/g), enhancing strength, durability, self-healing, and reducing permeability. SEM revealed that CBA exhibited high-intensity hydration peaks and calcite precipitates that filled voids, with calcite particles in the Raman spectra. The bio-admixture's hydrophobic treatment significantly improved the bonding with the binder, contributing to the concrete's enhanced performance. © 2025 Elsevier B.V., All rights reserved.