This study analyzed 400 well water samples from Akure, Nigeria, to assess the presence of Escherichia coli (E. coli), its antibiotic resistance, and plasmid profiles. The findings indicate that 19% of samples collected during the dry season and 27% during the rainy season were contaminated with E. coli, showing significant antibiotic resistance, especially to amoxicillin and sulfamethoxazole. Overall, the microbiological quality of well water in the area does not meet safety standards, posing severe health risks to the community.