Introduction:Periodontal infections, including gingivitis and periodontitis, form a major group among the most encountered chronic diseases with infective etiologies. Microorganisms present in gingival sulcus around teeth form microbial biofilm, which is most important cause of periodontal diseases. Biofilm, a three-dimensional (3D) microbial structure with cells enclosed within a self-produced extracellular matrix that may be attached to a substratum comprises the structure of a biofilm. This study aims to detect biofilm in microorganisms isolated from periodontal pockets and establishment of relation between biofilm with tobacco chewing and comorbidies. Material and methods:Total 100 Patients samples were collected using bent swab from periodontal lesions. Samples were processed aerobically and identification of the isolates were done along with simultaneous demonstration of in vitro biofilm formation. Results:Biofilm production was detected by using pre sterilized 96 well polystyrene micro titer plates. 71 samples were shown growth of microorganisms like Streptococcus viridians (36), Klebsiella pneumoniae (21), E. coli (6), Klebsiella oxytoca (4), Acinetobacter baumannii (1), Pseudomonas aeruginosa (1), Staphylococcus aureus (1), and Coagulase negative staphylococcus (1).19 isolates of Streptococcus viridians had formed biofilm out of 36 isolates. 16 isolates of Klebsiella pneumoniae had formed biofilm out of 21 isolates. 3 isolates of Klebsiella oxytoca had formed biofilm out of total 4 isolates. 2 isolates of E. coli had formed biofilm out of 6 isolates. One isolate, each of Acinetobacter baumannii, CONS and Pseudomonas aeroginosahad formed biofilm. Discussion: Out of 43 positive oral biofilms, 21% were tobacco chewers and out of negative oral biofilm, 15% were tobacco chewers. Among positive oral biofilms, 19% had co morbidities and among negative oral biofilm, 15% had co morbidity. Conclusion:The oral colonization by biofilm producing strains can also increase the risk of their dissemination to various human tissues and organs. Apart from that, biofilms cause resistance to many antimicrobial agents.