Chronic diabetic foot is a global burden affecting millions of people, and the chronicity of an ulcer is directly linked to the diverse bacterial burden and its biofilm mode of infection. The bacterial diversity of 100 chronic diabetic ulcer samples was profiled via traditional culturing method as well as metagenomic approach by sequencing the 16S rRNA V3 hyper-variable region on Illumina Miseq Platform (Illumina, Inc., San Diego, CA). All the relevant clinical metadata, including duration of diabetes, grade of ulcer, presence of neuropathy, and glycaemic level, were noted and correlated with the microbiota. The occurrence and establishment of bacterial biofilm over chronic wound tissues was revealed by Fluorescent in situ Hybridization and Scanning Electron Microscopy. The biofilm-forming ability of predominant bacterial isolates was studied via crystal violet assay and Confocal Laser Scanning Microscopy. The dominant phyla obtained from bacterial diversity analysis were Firmicutes, Proteobacteria, and Actinobacteria. The dominant aerobic pathogens identified by culture method are Pseudomonas, Proteus, Enterococcus, and Staphylococcus, whereas high-throughput sequencing revealed heightened levels of Streptococcus and Corynebacterium along with 22 different obligate anaerobes. The biofilm occurrence in chronic diabetic ulcer infection is well analysed. Herein, we illustrate the comprehensive pattern of bacterial infection and identify the community composition of chronic wound pathogenic biofilm.