This research aimed to identify bacterial species present in six detoriated painted relief samples collected from the catacomb of Kom Al-Shoqafa cemetery in Alexandria, Egypt, and to evaluate their antibacterial susceptibility toward chitosan nanoparticles synthesized in the study. Genomic DNA from three bacterial isolates were amplified using universal 16S rDNA primers and comparative 16S rDNA sequence analysis showed that the isolates had a sequence similarity of �91% with Paenibacillus albidus Paenibacillus typhi and Paenibacillus wynii The 16S rDNA gene sequence of these isolates were deposited in the GenBank database under accession numbers MW644543, MW644573, and MW644586, respectively. This research provides new molecular information on the microbial diversity of ancient detoriated painting samples in Egypt. Furthermore, the antibacterial susceptibility of these three bacterial strains were tested against synthesized chitosan nanoparticles (characterized by transmission electron microscopy) employing the well diffusion method, with the nanoparticles exhibiting significant antibacterial activities. Overall, this study provides valuable information on bacterial diversity in ancient painting samples and highlights chitosan nanoparticles as potential antibacterial agents that could be implemented for the preservation of these paintings.