Background <p>Antimicrobial resistance (AMR) is a growing global health concern, increasingly recognized to emerge not only from clinical and agricultural sources but also from natural and historical environments. Despite their ecological and cultural significance, ancient water bodies such as stepwells remain largely unexplored in the context of environmental resistomes.</p> Method <p>This study investigates AMR in Gandhak-ki-Baoli, an ancient sulfur-rich stepwell located in Delhi, India. A combined methodological approach involving culture-based microbial isolation and metagenomics sequencing was used to identify bacterial taxa and associated antibiotic resistance genes (ARGs).</p> Results <p>The analysis revealed a diverse microbial community harboring ARGs, including those conferring multidrug resistance. Several genes showed evidence of co-selection mechanisms with heavy metals and biocides. The stepwell’s unique environmental conditions characterized by stagnant water, low light, variable moisture, and limited nutrients that likely contribute to the persistence and potential horizontal transfer of resistance traits.</p> Conclusions <p>This is the first study to profile AMR in a historical stepwell, revealing the presence of a complex environmental resistome. The findings suggest that ancient water structures like stepwells can act as hidden reservoirs of AMR. These insights highlight the need to include such environments in future AMR surveillance efforts to better understand the broader ecological landscape of resistance.</p> Graphical Abstract <p></p>