Diarrheagenic Escherichia coli (DEC) are associated with frequent incidences of waterborne infections and pose health risk to individuals who contact water for domestic or recreational uses. Detection of DEC pathotypes in drinking water can be used as an indicator of fecal contamination. This study aimed to investigate the occurrence of DEC pathotypes and their capacity to form biofilms in drinking water samples collected from different water sources. In this study, PCR analysis was used to determine the occurrence of four clinically significant virulence genes of diarrheagenic E. coli, eaeA (Enteropathogenic E. coli), stx1, stx2 (Enterohemorrhagic E. coli) and sth (Enterotoxigenic E. coli), in drinking water samples (n = 35) by using specific primers and conditions. PCR amplicons were visualized by using agarose gel electrophoresis. A total of 12/35 (34%) samples were detected as positive for at least one of the four DEC virulence genes and 11/12 (91%) E. coli isolates harbored virulence gene while 1/12 (8%) E. coli isolates harbored none. The eaeA and sth genes were the most detected genes (75%), while stx1 and stx2 genes were least detected genes (66%). Biofilm assay confirmed that ETEC pathotypes can cause damage in enteric walls by attaching and effacing to persist diarrheal conditions. This study indicated that drinking water of different sources is contaminated with potential DEC pathotypes and it can be a source of diarrheal diseases. The amplification of four virulence genes associated with DEC pathotypes (EPEC, EHEC and ETEC) in drinking water demonstrates that potentially virulent DEC pathotypes are distributed in water sources and may be a cause of health concern. There is, therefore, an urgent need to monitor DEC pathotypes in drinking water.