This study evaluates the electrical potential and chemical alterations in laboratory-induced colistin-resistant Klebsiella pneumoniae, as compared to the susceptible strain using spectroscopic analyses. The minimal inhibitory concentration (MIC) of colistin, ζ-potential and chemical composition analysis of K. pneumoniae strains are determined. The results obtained for the K. pneumoniaeCol-R with induced high-level colistin resistance (MIC = 16.0 ± 0.0 mg/L) are compared with the K. pneumoniaeCol-S strain susceptible to colistin (MIC = 0.25 ± 0.0 mg/L). Fourier transform infrared (FTIR) and Raman spectroscopic studies revealed differences in bacterial cell wall structures and lipopolysaccharide (LPS) of K. pneumoniaeCol-R and K. pneumoniaeCol-S strains. In the beginning, we assumed that the obtained results could relate to a negative charge of the bacterial surface and different electrostatic interactions with cationic antibiotic molecules, reducing the affinity of colistin and leading to its lower penetration into K. pneumoniaeCol-R cell. However, no significant differences in the ζ-potential between the K. pneumoniaeCol-R and K. pneumoniaeCol-S strains are noticed. In conclusion, this mechanism is most probably associated with recognisable changes in the chemical composition of the K. pneumoniaeCol-R cell wall (especially in LPS) when compared to the susceptible strain.