The current study aimed to investigate the viability of biofilm formation klebsilla pneumoniae and Staphylococcus aureus. 440 urine samples were collected from patients suffering from urinary tract infection (UTI) from those who were admitted and visitors to Al-Ramadi Teaching Hospital, Al-Yarmouk Teaching Hospital, Al-Ramadi Teaching Hospital for women and children and , Teaching Laboratories in the Medical City for both genders for a period extended from 5 July, 2017 to 10 October, 2017. Samples were diagnosed by culturing them on a selective media and by biochemical testes , also, diagnosis was ensured by using VITEK-2 compact system. Results showed that K.pneumoniae isolation ratio was 17.1%(68) and S.aureus ratio was 13.1%(52). Their sensitivity was tested against 10 antibiotics, isolates showed uneven resistance according to the type of bacteria. Bacterial biofilm formation viability was detected by qualitative and quantitative methods, isolates per bacteria. Artificial material was used proteinase K and α-Amylase with triple concentrations to each enzyme 33,100,250 µg/ml and 20,100,200 mg/ml respectively, with biofilm inhibition of testing bacteria by precision calibration dishes method. Result showed noticeable decreasing in biofilm formation after enzymes addition in comparison with its formation before enzymes addition, where high decreasing ratio of biofilm according to proteinase K was at concentration 33µg/ml with 95% ratio, while less inhibition to K.pneumoniae, the same concentration showed inhibition viability at 93% and less inhibition viability at 259 µg/ml with 61%. Also, regarding to α-Amylase showed a high inhibition activity at 95%with concentration 100 mg/ml to S.aureus and less inhibition ratio was found at 20 mg/ml with 87%. While to K.pneumoniae , the triple concentrations showed inhibition activity ranged between 36-77%. Statistical analysis showed presence of significant variation between each enzyme concentration to both types of bacteria, also presence of significant variation among different concentration to each bacterial type at (P≤0.01).