Purpose: To characterize biofilm-forming capacity, extracellular polymeric substance (EPS), and antibiotic susceptibility (AST) of bacteria isolated from contact lenses (CL) and their accessories. Methods: Bacterial isolates from 20 CLs and their cases were evaluated for biofilm formation using crystal violet (CV) staining in tube and tissue culture plate assays. AST was performed by the Kirby-Bauer method. EPS production was assessed using Congo red agar (CRA) and Congo red broth (CRB), while carbohydrate, protein, and extracellular DNA (eDNA) contents were quantified. Results: The bacterial isolates included Achromobacter xylosoxidans, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus cohnii, Staphylococcus epidermidis, and Staphylococcus saprophyticus, identified and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Out of 110 isolates, biofilm production by CV staining showed 28.5% strongly positive, 38.1% moderately positive, and 23.3% weakly positive by the tube method, and 32.1%, 18.6%, and 29.0%, respectively by tissue culture plate method; 14.3% were non-biofilm producers. The selected isolates were susceptible to the tested antibiotics, but resistance was noted in A. xylosoxidans. The EPS production was confirmed by black coloration in CRA plates, and CRB tubes with carbohydrate content was found to increase, whereas protein content was slightly reduced, and the presence of eDNA was detected after 48 hours. The scanning electron microscopy images depicted the presence of branched and sheet-like extracellular matrix structures. Physiological conditions like acidic pH and the presence of ribose sugar showed reduced biofilm; conversely, incubation at 28 °C enhanced bacterial attachment. In Staphylococcus species, increased salt concentration supported biofilm formation. Conclusions This study showed that bacteria isolated from CL of asymptomatic users had significant biofilm-forming ability. A. xylosoxidans and CoNs Staphylococcus were the predominant biofilm producers. CRB was an effective qualitative method for detecting EPS. These findings indicate bacterial colonization rather than infection and highlight the importance of proper contact lens hygiene and preventive care.