Background: [99mTc]Tilmanocept, a novel CD206 receptor-targeted radiopharmaceutical, was evaluated in an open-label, phase III trial to determine the false negative rate (FNR) of sentinel lymph node biopsy (SLNB) relative to the pathologic nodal status in patients with intraoral or cutaneous head and neck squamous cell carcinoma (HNSCC) undergoing tumor resection, SLNB, and planned elective neck dissection (END). Negative predictive value (NPV), overall accuracy of SLNB, and the impact of radiopharmaceutical injection timing relative to surgery were assessed. Methods and Findings: This multicenter, non-randomized, single-arm trial (ClinicalTrials.gov identifier NCT00911326) enrolled 101 patients with T1–T4, N0, and M0 HNSCC. Patients received 50 µg [99mTc]tilmanocept radiolabeled with either 0.5 mCi (same day) or 2.0 mCi (next day), followed by lymphoscintigraphy, SLNB, and END. All excised tissues were evaluated for tissue type and tumor presence. [99mTc]Tilmanocept identified one or more SLNs in 81 of 83 patients (97.6 %). Of 39 patients identified with any tumor-positive nodes (SLN or non-SLN), one patient had a single tumor-positive non-SLN in whom all SLNs were tumor-negative, yielding an FNR of 2.56 %; NPV was 97.8 % and overall accuracy was 98.8 %. No significant differences were observed between same-day and next-day procedures. Conclusions: Use of receptor-targeted [99mTc]tilmanocept for lymphatic mapping allows for a high rate of SLN identification in patients with intraoral and cutaneous HNSCC. SLNB employing [99mTc]tilmanocept accurately predicts the pathologic nodal status of intraoral HNSCC patients with low FNR, high NPV, and high overall accuracy. The use of [99mTc]tilmanocept for SLNB in select patients may be appropriate and may obviate the need to perform more extensive procedures such as END.