Good health and well-being are among the UN Sustainable Development Goals. Based on this, the present study aimed to investigate a prevalent disease in Iraq, namely gallstone disease, along with the microbial infections associated with cholelithiasis and their antibiotic sensitivity patterns. The study was conducted at Teaching al-Sadr Hospital and Al-Zahrawi Surgical Hospital in Maysan, Iraq, from November 2021 to December 2022. Sixty cases of cholelithiasis in men and women between the ages of 20 and 50, who underwent cholecystectomy, were selected. The most common organism identified was Escherichia coli, found in 20 patients (33%). This was followed by Pseudomonas fluorescens in 7 patients (11.6%), Klebsiella pneumoniae in 6 patients (10%), Serratia marcescens in 6 patients (10%), Pseudomonas aeruginosa in 5 patients (8.3%), Kluyvera cryocrescens in 5 patients (8.3%), Proteus vulgaris in 5 patients (8.3%), Raoultella orithinolytica in 3 patients (5%), Klebsiella oxytoca in 2 patients (3.3%), and Enterobacter cloacae in 1 patient (1.6%). The biliary organisms showed various antibiotic sensitivity patterns. The highest sensitivity was observed with Cefotaxime (71%). The other antibiotics arranged as following; Amikacin (56%), Gentamicin (50%), Ciprofloxacin (46%), Meropenem (40%), Ceftazidime (35%), Imipenem (35%), Ertapenem (10%), Ampicillin (10%), Amoxicillin clavulanate (10%), and Cefepime (8%). In addition, female gender and advanced age were noted as contributing factors to gallstones. Cholesterol stones were found to be more common than other kinds of gallstones.