Two hundred fifty mid-stream urine specimens were collected from Baqubah Teaching Hospital and Al-Batool Teaching Hospital from patients with urinary tract infections (UTI). Of these investigated urine specimens, 66 (26.4%) specimens showed positive growth culture of Gram-negative bacteria. From these, Escherichia coli was the most prevalent bacteria of the examined culture (41, 62.12%). Additionally, the cup assay was used to determine colicin producers while the most efficient colicin producers were estimated by the formation of larger inhibition zone. Approximately half of the investigated E. coli isolates (20, 49 %) was colicin producers. Colicins was extracted after induction by mitomycin-C showed a concentration of 3020 μg/ml,  as estimated utilizing the Lowry method, while its activity was 80 U/ml. Our study results showed that colicin had significant antibiofilm activity (P≤ 0.05) against Candida albicans and the effect seemed to be concentration dependent. . However, the values of biofilm inhibition varied depending on the different tested isolates. The biofilm of isolate 5 showed the most significant inhibition (P≤ 0.05) by colicin with a value of 46%, while isolate 3 was less affected with an inhibition rate of 19% at the concentration of 2500 μl/ml.