Abstract Purpose Sentinel lymph node (SLN) biopsy has proven to reliably stage the clinically negative neck in early-stage oral squamous cell carcinoma (OSCC). [^99mTc]Tc-tilmanocept may be of benefit in OSCC with complex lymphatic drainage patterns and close spatial relation to SLNs. Methods A prospective within-patient evaluation study was designed to compare [^99mTc]Tc-tilmanocept with [^99mTc]Tc-nanocolloid for SLN detection. A total of 20 patients with early-stage OSCC were included, who underwent lymphoscintigraphy with both tracers. Both lymphoscintigraphic images of each patient were evaluated for SLN detection and radiotracer distribution at 2–4 h post-injection. Results The injection site’s remaining radioactivity was significantly lower for [^99mTc]Tc-tilmanocept (29.9%), compared with [^99mTc]Tc-nanocolloid (60.9%; p < 0.001). Radioactive uptake in SLNs was significantly lower for [^99mTc]Tc-tilmanocept (1.95%) compared with [^99mTc]Tc-nanocolloid (3.16%; p = 0.010). No significant difference was seen in SLN to injection site ratio in radioactivity between [^99mTc]Tc-tilmanocept (0.066) and [^99mTc]Tc-nanocolloid (0.054; p = 0.232). A median of 3.0 and 2.5 SLNs were identified with [^99mTc]Tc-tilmanocept and [^99mTc]Tc-nanocolloid, respectively (p = 0.297). Radioactive uptake in higher echelon nodes was not significantly different between [^99mTc]Tc-tilmanocept (0.57%) and [^99mTc]Tc-nanocolloid (0.86%) (p = 0.052). A median of 2.0 and 2.5 higher echelon nodes was identified with [^99mTc]Tc-tilmanocept and [^99mTc]Tc-nanocolloid, respectively (p = 0.083). Conclusion [^99mTc]Tc-tilmanocept had a higher injection site clearance, but at the same time a lower uptake in the SLN, resulting in an SLN to injection site ratio, which was not significantly different from [^99mTc]Tc-nanocolloid. The relatively low-radioactive uptake in SLNs of [^99mTc]Tc-tilmanocept may limit intraoperative detection of SLNs, but can be overcome by a higher injection dose.