We aimed to develop a multiplex PCR (mPCR) assay utilizing the afaC gene for the detection of DAEC pathotypes and the bla_TEM gene for the identification of ESBL production in clinical E. coli isolates along with an Internal Amplification Control (IAC) to rule out false negative test results. Monoplex PCR assays were first established for the afaC gene and bla_TEM gene using 60 characterized E. coli isolates from various clinical samples. Subsequently, an mPCR assay was designed to detect both the genes simultaneously along with an IAC to rule out false negative reactions. The effectiveness of this assay was validated using 80 additional clinical isolates. The overall occurrence of DAEC in the study was found to be 0.7% (1/140). ESBL production was detected in 40.7% of the tested isolates, indicating a concerning prevalence of drug-resistant strains. This study emphasizes the value of an in-house mPCR assay as a crucial tool for simultaneously identifying DAEC and ESBL E. coli strains. The inclusion of an IAC in the PCR protocol bolstered the assay’s reliability. This innovation offers a vital resource for effective infection management and contributes to the comprehension of pathogenicity and resistance mechanisms in clinical E. coli isolates.