Gram-negative folliculitis (GNF) is an infection that is seen as a complication in patients with acne vulgaris and is consequent to long term received systemic antibiotics. Two main types have been described; type-I is around 80% of cases with the presence of multiple papules and pustules and type-II occurs in 20% of cases with the characterization of inflammation of nodules or cyst. Objective of current study is to characterize the gram-negative bacteria from GNF lesions and evaluate the antimicrobial susceptibility against some of the commercially available antibiotics. The confirmatory characterization of isolated bacteria has been performed, using 16S rRNA sequencing and broth dilution assay has been performed to evaluate minimum inhibitory concentration (MIC) of antibiotics against isolated gram negative bacteria. There are 21 different gram-negative bacterial strains have been characterized and submitted to NCBI which mainly contains Firmicutes and Proteobacteria phylum primarily belong to the descriptions of all isolated gram-negative bacteria from acne lesions. The abundance of family bacteria of Enterobacteriaceae, Moraxellaceae and Paenibacillaceae was 77%, 19% and 4% respectively. Klebsiella , Enterobacter and Acinetobacter demonstrated resistance to erythromycin, zinc oxide, azelaic acid, nadifloxacin, and gentamicin, while tetracycline and doxycycline tolerance was shown only by E. liudwigii. The current study shows that Klebsiella pneumoniae, Enterobacter sp., Proteus mirabilis and Acinetobacter sp. bacterial strains may play a significant role in the pathogenesis of Gram-negative folliculitis.