AimsThis study aimed to investigate the diversity and determinants of biofilm formation among clinical Acinetobacter baumannii Indian isolates and assess their relationship with antimicrobial resistance profiles, biofilm-associated genes, and genetic lineages revealed through whole-genome analysis.Methods and results230 A. baumannii clinical isolates across India (2015-2022) were tested for antibiotic susceptibility using the VITEK 2 system. Biofilm formation was quantified via the Tissue Culture Plate method. Whole genome sequencing (Illumina MiSeq) and bioinformatic analysis were performed to identify biofilm-associated genes, antimicrobial resistance genes and sequence types. Statistical associations were assessed using Kruskal-Wallis, Spearman's, and Fisher's tests. 85.22% of isolates were multidrug-resistant (MDR), and 100% exhibited biofilm formation, with 52.17% strong, 39.57% moderate, and 8.26% weak biofilm producers. Genes including ompA, bfmR, pgaA, pgaB, and pgaD were universally present. No significant association was observed between biofilm formation and antibiotic resistance (P = 0.55), specimen type (P = 0.54), or the presence of specific biofilm-related genes (P > 0.05). 21 sequence types (STs) were identified, with ST2 being the most prevalent (51.73%). Strong biofilm formation was more common in ST164, ST1, and ST575.ConclusionsThis study demonstrates a high prevalence of MDR and strong biofilm-forming A. baumannii isolates in India. Biofilm formation appeared independent of resistance or gene carriage but showed lineage-linked variation across sequence types.