Abstract Introduction It has been estimated by the National Institutes of Health, that more than 80% of persistent bacterial infections are likely to involve biofilms. Medically relevant biofilms are gaining interest in a number of diseases, as they seem to be a key component in resistant bacterial colonization. The aim of the present work was to estimate the prevalence of biofilms formed by different bacterial isolates in different clinical and Health-care associated infections in some of Egyptian hospitals. Patients and methods: Specimens taken from 173 patients at different hospitals. Qualitative and quantitative assessment of biofilm formation were determined by using visual tube and spectrophotometric methods respectively under the effect of different chemical and physical factors on biofilm formation. Bacterial isolates were subjected to antimicrobial sensitivity test. Results: One hundred and sixty three microbial isolates were recovered. Biofilm formation was detected in 60 (70.6%) by using visual tube method. The result showed that difference in media components play role in providing biofilm formation. By using spectrophotometric method, biofilm formation was detected in 20 (23.5%). The highest percentage of biofilm forming isolates was obtained at 30°C/24hrs as 70.6% were biofilm producers Bacterial isolates were subjected to antimicrobial sensitivity test. In case of biofilm forming isolates, 63.4% were resistant to cefotaxime and 40.0% were resistant to imipenem. Conclusion: It concluded that, those techniques previously mentioned are of great value in revealing the role of adherence, colonization and biofilm formation in pathogenicity of microorganisms.