Background: Gallstone disease (GSD) is a significant global health burden with variable prevalence influenced by metabolic, genetic, and infectious factors. Increasing evidence suggests that Gram-positive bacteria, particularly Staphylococcus aureus and Enterococcus species, contribute to gallstone pathogenesis through enzymatic activity and biofilm formation. Objectives: To characterize Gram-positive bacteria within gallstones from Iraqi patients, evaluate their biofilm-forming capacity, and analyze the relationship between bacterial colonization, gallstone type, and cholesterol levels. Methods: A total of 100 gallstones were obtained from patients undergoing elective cholecystectomy between October 2024 and March 2025. Stones were aseptically processed for bacterial isolation and identification using selective culture media and the VITEK® 2 Compact System. Serum cholesterol levels were determined by enzymatic colorimetric assay. Biofilm formation was quantified via the 96-well microtiter plate method, and statistical correlations between gallstone type, cholesterol level, and bacterial presence were analyzed. Results: Cholesterol stones (57%) were more prevalent than pigment (40%) and mixed stones (3%). Bacterial growth was observed in 43% of gallstones, with Enterococcus species (31 isolates) predominating over S. aureus (12 isolates). Species-level identification revealed E. faecalis (n= 16), E. faecium (n= 9), and E. gallinarum (n= 6), marking the first reported isolation of E. gallinarum from gallstones. Cholesterol concentrations were significantly higher in sterile stones (median 235 mg/dL) compared to bacteria-positive stones (173-186 mg/dL) (P < 0.0001). Biofilm analysis showed all S. aureus isolates as strong producers, whereas E. faecalis exhibited predominantly moderate-to-strong formation, while E. faecium and E. gallinarum displayed weaker capacities. Conclusion: A significant interplay between microbial colonization and gallstone composition. Strong biofilm-producing Gram-positive bacteria, particularly S. aureus and E. faecalis, may facilitate stone maturation and persistence. The novel isolation of E. gallinarum expands the spectrum of biliary microbiota.