ABSTRACT This study investigated the presence of adenoviral markers (Library-Independent microbial source tracking tool) in the groundwater of Ludhiana, Punjab, India, a hitherto unexplored area. While deep aquifers post-chlorination were adenovirus free, shallow aquifers near farm wastewater pits exhibited human adenovirus and bovine adenovirus. Coliform-negative samples also harboured pathogens, highlighting the limitations of conventional indicators. Surface water displayed higher viral contamination, potentially impacting groundwater. The use of farm wastewater for irrigation and open pit disposal emerged as key contributors, advocating for sustainable wastewater management. Physiochemical and microbial analyses revealed variations across sites, emphasising regional and temporal variations. The weighted arithmetic water quality index ranged from good to very poor, with deep aquifers showing better quality than shallow ones. A novel approach incorporating graphical representations of adenovirus estimations alongside water quality index provided a more comprehensive understanding. Intriguingly, the study revealed the presence of coliforms irrespective of water quality grade, questioning its reliability as a sole indicator. Correlations between specific water quality grades and adenovirus types suggested targeted control measures. The lack of significant correlations between viral markers and conventional parameters in groundwater compared to surface water studies highlighted the unique dynamics of groundwater contamination.