This research aimed to assess the physicochemical and bacteriological quality of underground water, and determine the antibiotic susceptibility and presence of plasmids in multiple antibiotic-resistant (MAR) Escherichia coli O157:H7 in underground water sources in Ado-Ekiti. Physicochemical and bacteriological analysis of water samples were carried out using standard methods, an antibiotic susceptibility test was investigated using the standard disc diffusion method and plasmid analysis of MAR isolates was carried out using the polymerase chain reaction (PCR) technique. The physicochemical parameters analyzed were within WHO recommendations except for pH and potassium while the water samples did not conform to the WHO bacteriological recommendations for drinking water. A total of 272 E. coli were isolated and identified, among which 150 isolates were non-sorbitol fermenters (NSF) and taken as presumptive E. coli O157. MAR to three and more classes of antibiotics used were observed among these NSF with high MAR-Index, >0.2. Plasmid analysis of selected 15 isolates among the MAR NSF showed that their resistance to antibiotics was likely plasmid-mediated as they carry one to two plasmids on them. The study revealed that the water samples from Ado-Ekiti metropolis are unsafe for consumption.