Background: Thyroid fine-needle aspiration biopsy (FNAB) is a commonly used diagnostic procedure in patients with suspected thyroid cancer; however, it may induce pain, anxiety, and fear during the procedure. Objective: This open-label randomized controlled trial aimed to evaluate the effect of virtual reality (VR) on pain as the primary outcome and anxiety and fear of pain as secondary outcomes in patients undergoing thyroid FNAB. Methods: The study was conducted between January 19, 2025, and April 30, 2025, at Gaziantep City Hospital, Türkiye. A total of 100 patients with suspected thyroid nodules were randomly assigned to either a VR intervention group (n=50) or a control group (n=50). Data were collected using a patient information form, the visual analog scale (VAS), the Beck Anxiety Inventory (BAI), and the Fear of Pain Questionnaire-III (FPQ-III). Results: After adjustment for baseline pain, previous thyroid mass diagnosis, and voice tone changes, the VR group had statistically significantly lower postintervention pain scores than the control group (adjusted mean 3.627 vs 4.493; F_1,95=4.021; P=.048; partial η²=0.041). However, the unadjusted between-group comparison for pain was not statistically significant (P=.12), and the unadjusted effect size was small, with a 95% CI that crossed 0 (Cohen d=−0.33, 95% CI −0.72 to 0.07). No statistically significant adjusted between-group differences were observed for anxiety (P=.48) or fear of pain (P=.07). Unadjusted standardized between-group effect sizes were also small for anxiety (d=−0.17) and fear of pain (d=−0.11). Conclusions: The adjusted analysis suggested a small reduction in procedural pain with VR; however, the between-group difference was not statistically significant in the unadjusted analysis and reached statistical significance only after adjustment for baseline pain and 2 nonprespecified covariates selected on the basis of observed baseline imbalance. Moreover, the observed adjusted effect (f=0.207) was smaller than the minimum effect size the trial was powered to detect (f=0.283). No statistically significant adjusted between-group effects were found for anxiety or fear of pain. Therefore, the potential analgesic effect of VR should be interpreted cautiously and confirmed in larger, adequately powered trials. Trial Registration: ClinicalTrials.gov NCT06792929; https://clinicaltrials.gov/study/NCT06792929