Abstract : Dental biofilm associated periodontal disease within the orofacial cleft anomalies are the leading oral health issue in recent decades. Appropriate diagnosis and novel strategic approach with respect to treatment has been of current needs in order to develop patient healthy without consequences. As culture-based techniques found to be not suitable in identifying the oral biofilm producers, current study meticulously executed with 16S rRNA gene sequencing to provide detailed insight towards oral biofilm producers of orofacial cleft cases. Samples were collected from cleftpalate, cleft lip and both lip and palate irrespective to the age, sex and races reported in different microbiology labs. Biofilm growth was determined after 24 or 48 hrs of incubation by measuring the optical density using spectrophotometer. The v3-v4 hyper variable regions of the 16S ribosomal RNA genes from the oral cavity samples were amplified via PCR. The libraries were subjected to sequencing with an Illumina MiSeq sequencing platform using MiSeq Pair-end 300 methods. Phylogenetic analysis of 16S rRNA sequences significantly identified three strains such as Citrobacter tructae 43(23.11%) Enterobacter cloacae 39 (20.96%) and Klebsiella michihanensis 37(19.89 %) hereby finding their involvement was seems to be the first has been of prime interest in association with periodontal disease in orofacial cleft patients. Study results hypothesized that these strains could have been entered into orofacial cleft cases through contaminated tooth brush while using common bathrooms. Moreover, these strains are notorious biofilm producer and this cause the highest number of acute and chronic dental biofilm associated periodontal disease.