Purpose <p>Fever of unknown origin (FUO) is a prolonged fever with unknown etiology after an extensive outpatient or inpatient workup. ¹⁸F-Flurodeoxyglucose (FDG) positron emission tomography (PET) with computed tomography (CT) has shown a higher diagnostic yield than CT. This study assesses the diagnostic yield of ¹⁸F-FDG PET/CT-guided targeted biopsy in FUO.</p> Methods <p>This single-centre retrospective study included FUO patients who underwent ¹⁸F-FDG PET/CT-guided biopsy. A distinct focal area of FDG-uptake above the surrounding background within the organ of interest was considered PET-positive and targeted using automated robotic arm–assisted PET/CT-guided biopsy systems. Procedure-related complications were recorded, and histopathological examination was taken as the reference standard. Diagnostic yield and other PET-based parameters were calculated.</p> Results <p>Data from 165 FUO patients [median age 47 years (IQR, 30–60)] who underwent ¹⁸F-FDG PET/CT-guided biopsy between 2017 and June 2025 were analysed. 64 (39%) had undergone a previous procedure with negative or inconclusive results. The most common site of targeted biopsy was the lungs (71/165, 43%). The diagnostic yield of ¹⁸F-FDG PET/CT-guided biopsy in FUO patients was 96.4% (159/165), with the most common cause being malignancy (70/165, 42%), followed by infection (58/165, 35%), autoimmune (16/165, 10%) and inflammatory (7/165, 4%) causes. The median SUVmax of the target lesions was 11.8 (IQR, 8.6–16.3; range, 3.3–53.8) with no significant difference among target lesions of different etiologies (<i>p</i> = 0.412). Only six patients (3.6%) had procedure-related complications.</p> Conclusion <p>Robotic arm-assisted ¹⁸F-FDG PET/CT-guided biopsy is a safe and accurate procedure in FUO patients having FDG-avid accessible lesions and may help in early diagnosis and management.</p>