Microbial contamination of water sources remains a serious problem in the Kathmandu Valley, Nepal. This study was conducted to comprehensively investigate the occurrence of pathogenic bacteria in water samples collected from the valley, most of which were used for drinking and other domestic purposes. The water samples consisted of 10 samples of commercial jar water, 10 of groundwater from shallow dug wells, 3 of groundwater from deep tube wells, 1 of stone spout water, 2 of spring water, and 2 of river water. DNA microarray analysis targeting 941 different pathogenic bacterial species/groups showed the prevalence of Acinetobacter, Arthrobacter, Brevibacterium, Pseudomonas, and Legionella and the presence of biosafety level 3 pathogens in some samples, which were partially confirmed by genus-specific PCR quantification for Legionella and Brucella. Additionally, the occurrence of most pathogens detected via DNA microarray analysis was not correlated with that of fecal indicator bacteria (total coliforms and Escherichia coli). These results will aid in understanding the overall picture of pathogenic bacterial contamination and in screening important pathogens that should be investigated in detail.