Fusobacterium nucleatum has been increasingly implicated as a causative agent of various diseases, such as inflammatory bowel disease. Moreover, the gastrointestinal tracts of patients with colorectal cancer (CRC) also have been shown to be colonized by this bacterium. We aimed to determine the prevalence of F. nucleatum among CRC and non-CRC Iranian patients and to investigate potential associations between fadA-positive F. nucleatum and diagnosed CRC cases. Eighty patients admitted to two main hospitals in Tehran, Iran, were enrolled. The patients were aged between 20 and 75 and were diagnosed by a gastroenterologist. A trained surgeon used standard surgical protocols to collect two CRC biopsy samples per patient. One of the samples was used for pathologic examination, and the other was subjected to DNA extraction and PCR. Lesion colonization by F. nucleatum and expression of its major virulence factor, fadA, were investigated. The fadA-positive F. nucleatum strain was absent in all the lesions obtained from non-CRC patients. All patients with lesions that were colonized with fadA-positive F. nucleatum were diagnosed as CRC (p < 0.05); selected patients were sent for further intensive treatment. We found a significant association between the presence of F. nucleatum colonization and lesions from CRC patients (p 0.0001; odds ratio, 6.74; 95% confidence interval, 2.5-18.07). Our study confirmed colonization of the fadA-positive F. nucleatum on lesions from 80 Iranian CRC patients. New therapeutic strategies to achieve eradication of F. nucleatum are necessary for clinical management of patients suspected of having or prone to developing CRC.