Background: Enterococcus faecalis is a causative agent for urinary tract infections (UTIs) in Iraq and worldwide, even though it is a commensal bacterium in human and animal intestines. It can cause different illnesses due to its ability to produce various virulence factors. A pore-forming toxin (cytolysin) is the most virulence factor in this bacterium. Objective: This study aims to molecularly investigate the frequency of cytolysin toxin among E. faecalis isolated from UTIs. Methods: A hundred and eighty urine specimens were collected from females diagnosed with UTIs. Traditional laboratory and molecular methods were used for bacterial identification and toxin detection using a modified DNA extraction method. Results:  The findings revealed that 27.7% (50\180) of causative agents in UTIs were E. faecalis based on the molecular technique that targeted a housekeeping gene (ddI) with specific primers using polymerase chain reaction (PCR). Most of the isolates harboured the cytolysin toxin gene (cylL_L) with a frequency rate of 92% (46\50). Conclusions: A considerable prevalence of cytolysin-positive isolates in UTIs, which is a worrying indicates of the extensive spreading of a toxic strain in UTIs. The modified method for DNA extraction in gene detection was successfully used to amplify a housekeeping gene (ddI) and a virulence gene (cylL_L) for cytolysin toxin detection, and this approach can be utilised for rapid bacterial identification and gene detection in medical and research purposes with a large sample size in an inexpensive manner within a short time.