Percutaneous ethanol injection (PEI) is an alternative therapy for thyroid nodules (TN). However, some concern is raised on its carcinogenic effects. To evaluate the cytological and clinical changes caused by PEI in patients with benign TN. Thirty-nine patients with TN (23.1% hyperfunctioning) were submitted to a median of three PEI sessions. After a median of 17 months, patients were reassessed. A new ultrasound-guided fine needle biopsy (US-FNB) was performed, and the smears were analyzed after May-Grünwald-Giemsa staining. The diagnostic findings and the cellular characteristics were compared before and after treatment. There was an increase in the proportion of nondiagnostic/unsatisfactory results (from 2.5% to 18.9%). No malignant cases were observed. The proportion of moderate/intense macrophage infiltration decreased from 60% to 15%. Before treatment, 23.1% patients had hyperthyroidism, which was completely or partially resolved in 66.7%. By ultrasound, the percentage of homogeneous nodules decreased from 64.0% to 38.4% (p=0.0235), and the mean nodule volume decreased from 13.4 ± 12.2 to 5.3 ± 5.1 cm(3). We demonstrate that PEI increases the proportion of nondiagnostic/unsatisfactory results from US-FNB. Therefore, cytological findings after PEI must be evaluated with caution. Our results also suggest that PEI is an efficacious and safe therapeutic option, with no carcinogenic effects observed on cytological evaluations. Safety and efficacy must be evaluated in larger studies with longer follow-up periods.