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Abstract Background Two-dimensional (2D) ultrasound is well established for thyroid nodule assessment and treatment guidance. However, it is hampered by a limited field of view and observer variability that may lead to inaccurate nodule classification and treatment. To cope with these limitations, we investigated the use of real-time three-dimensional (3D) ultrasound to improve the accuracy of volume estimation and needle placement during radiofrequency ablation. We assess a new 3D matrix transducer for nodule volume estimation and image-guided radiofrequency ablation. Methods Thirty thyroid nodule phantoms with thermochromic dye underwent volume estimation and ablation guided by a 2D linear and 3D mechanically-swept array and a 3D matrix transducer. Results The 3D matrix nodule volume estimations had a lower median difference with the ground truth (0.4 mL) compared to the standard 2D approach (2.2 mL, p < 0.001) and mechanically swept 3D transducer (2.0 mL, p = 0.016). The 3D matrix-guided ablation resulted in a similar nodule ablation coverage when compared to 2D-guidance (76.7% versus 80.8%, p = 0.542). The 3D mechanically swept transducer performed worse (60.1%, p = 0.015). However, 3D matrix and 2D guidance ablations lead to a larger ablated volume outside the nodule than 3D mechanically swept (5.1 mL, 4.2 mL (p = 0.274), 0.5 mL (p < 0.001), respectively). The 3D matrix and mechanically swept approaches were faster with 80 and 72.5 s/mL ablated than 2D with 105.5 s/mL ablated. Conclusions The 3D matrix transducer estimates volumes more accurately and can facilitate accurate needle placement while reducing procedure time.