Optimal management of nonsmall cell lung cancer (NSCLC) depends on tissue diagnosis and accurate staging. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is minimally invasive and provides cytological confirmation of malignant mediastinal disease. The aim was to assess the accuracy of EUS-FNA in cases of enlarged mediastinal lymphadenopathy (LN) of unknown aetiology and in the staging of NSCLC. A total of 52 consecutive patients with stage I-IIIb NSCLC or enlarged mediastinal LN of unknown aetiology underwent EUS-FNA. Negative results were confirmed with a surgical procedure: mediastinoscopy, video-assisted thoracic surgery (VATS) or lobectomy with systematic mediastinal lymph node dissection. In total, 34 patients had EUS-FNA performed for diagnosis, whilst the remaining 18 had EUS-FNA for staging. The overall sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy (95% confidence interval) were 93% (77-99), 100% (78-100), 100% (87-100), 88% (63-99) and 95% (84-99), respectively. When EUS-FNA was used in patients with NSCLC, the sensitivity, specificity, PPV, NPV and accuracy were 92% (73-99), 100% (69-100), 100% (85-100), 83% (51-98) and 94% (80-99), respectively. For mediastinal LN of unknown aetiology, no malignant disease was missed. Endoscopic ultrasound-guided fine-needle aspiration is an accurate tool for assessing mediastinal lymph node involvement in nonsmall cell lung cancer and in the diagnosis of unexplained mediastinal lymphadenopathy. Endoscopic ultrasound-guided fine-needle aspiration is a minimally invasive procedure that can be used as an adjunct or alternative to mediastinoscopy.