Radiofrequency ablation (RFA) can be a therapeutic option in medically inoperable lung cancer patients. In this study, we evaluated a prototype bipolar RFA device applicator that can be deployed from a standard endobronchial ultrasound (EBUS) bronchoscope to determine feasibility and histopathological analysis in animal models. Rabbit lung cancers were created by transbronchial injection of VX2 rabbit cancer cells. Once the tumors were developed, they were ablated transpleurally, under EBUS guidance using the prototype RFA device. The animals were then sacrificed for specimen resection. Pig inflammatory lung pseudo-tumors and lymphadenopathy were created by transbronchial injection of a talc paste and ablated transbronchially under EBUS guidance. Pigs were evaluated at five days, two weeks, and four weeks following ablation by bronchoscopy and cone-beam computed tomography before necropsy. Nicotinamide adenine dinucleotide hydrogen diaphorase staining was employed to measure the ablation area. Twenty-four VX2 rabbit tumors were ablated. The total ablated area ranged from 0.6 to 3.0 cm2 (mean: 1.8 cm2), corresponding to a total energy range of 1 to 6 kJ. Six pig lung pseudo-tumors and five mediastinal lymph nodes (LNs) were ablated. Adjacent airway ulceration was observed in three ablations of LNs. These airway complications resolved within four weeks of RFA without any treatment. There was no hemoptysis, air embolism, respiratory distress, or other serious complication noted. In these two animal models, we provide evidence that EBUS-guided bipolar RFA is feasible and histopathology shows that can ablate lung tumors and mediastinal lymph nodes under real-time ultrasound guidance.