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The increasing of multi-drug among pathogenic microbes is rendering antibiotics ineffective. Consequently, efforts are now concentrated on addressing this challenge through developing novel antibiotics and enhancing existing ones. This study combined ciprofloxacin (CIP) with titanium dioxide nanoparticles (TiO2NPs). We characterized the prepared nanoparticles (NPs) using several methods, including UV-Vis spectra, XRD, FESEM, TEM, and FTIR. The well diffusion agar was used to study the antibacterial activity of ciprofloxacin (CIP) alone and combined with titanium dioxide nanoparticles CIP@TiO2NPs. CIP@TiO2NPs showed higher antibacterial activity against Klebsiella pneumoniae (K.pneumoniae) and Streptococcus mutans S.mutans. The CIP@TiO2NPs showed remarkable inhibitory properties compared to CIP alone and TiO2NP alone, with its inhibition zone 28.50 ± 0.20 and 17.50 ± 0.10 in K.pneumoniae and S.mutans, respectively. Insilico study was done on bacterial strains to describe the effective binding behavior towards the ciprofloxacin@TiO2 adsorption system. The best conformers, from 50 conformational adsorption systems, were analyzed with a significant favorable inhibition with binding energy values of -9.61 kcal/mol and − 9.40 kcal/mol with K.pneumoniae and S. mutans, respectively. The interaction between CIP@TiO2NPs nanoparticles and Klebsiella pneumonia (ID: 8JGW) was studied using 50 conformations. The results showed binding energies up to -9.61 kcal/mol, indicating high interaction efficacy. Compared to TiO2NPs and CIP alone, CIP@TiO2NPs displayed the highest antibacterial and anti-biofilm properties against pathogenic bacteria. CIP@TiO2NPs have demonstrated promising results, suggesting that they may prove to be a dependable treatment for K. pneumoniae and S.mutans in the future and a possible agent for reducing bacterial biofilm during bacterial infections.