Purpose: Thyroglobulin (Tg) assessment in the needle washout after fine-needle aspiration biopsy (FNAB) of a suspicious neck lymph node (LN) is known to improve the diagnostic accuracy in patients with papillary thyroid cancer (PTC). However, there is still controversy on the best diagnostic cut-off levels for FNAB-Tg and whether thyroglobulin antibody (TgAb) positivity affects FNAB-Tg. The objectives of this study were to determine (i) the diagnostic power of different cut-offs for FNAB-Tg and (ii) if serum TgAb(+) negatively affects the FNAB-Tg evaluation. Methods: This was a retrospective cohort study analyzing PTC patients with suspicious neck LNs, in a university hospital setting, from October 2009 to October 2013. In total, 103 patients with PTC (226 LNs) undergoing ultrasound-guided FNAB for LNs were included. Cytology and FNAB-Tg levels were compared in reference to LN histopathology and the effect of TgAb(+) on FNAB-Tg levels was evaluated. Results: The diagnostic accuracies of FNAB-Tg cut-off of 1 and 10 ng/mL were 94.1% and 88.2%, respectively. Raising the cut-off from 1 to 10 ng/mL led to decreased sensitivity rates (91.9% vs. 83.9%). The receiver operating characteristic curve analysis demonstrated that the best FNAB-Tg cut-off was 1.2 ng/mL. There were no LNs with an FNAB-Tg >= 10 ng/mL that turned out to be cytologically or histopathologically benign. FNAB-Tg levels of the histopathologically malignant LNs were similar between TgAb (+) and TgAb (-) patients (p = 0.546). Serum Tg predicted FNAB-Tg levels above 1 ng/mL (p = 0.002) and FNAB-Tg predicted malignant histopathology (p = 0.004), both independently of the TgAb status of the patient. Conclusions: FNAB-Tg >= 1 ng/mL has a superior diagnostic power, irrespective of TgAb (+), in PTC patients with suspected LN involvement.