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Epstein-Barr virus (EBV) is a common viral agent causing infectious mononucleosis and can present with fever, fatigue, pharyngitis and cervical lymphadenopathy. Isolated gallbladder wall thickening without leukocytosis or splenomegaly is an atypical presentation of EBV infection. We present a case of a young woman with 2–3 days of epigastric pain, nausea and vomiting with gallbladder wall thickening on imaging. Despite an initial diagnosis of acalculous cholecystitis, a hepatobiliary iminodiacetic acid scan did not reveal acute cholecystitis. Positive EBV serology suggested a viral cause. This case emphasizes the need for clinicians to broaden their differential when considering isolated gallbladder wall thickening. Early identification of a viral cause can reduce unnecessary invasive interventions such as cholecystectomy.