The aim of this first-time-in-human non-randomized dose-escalating prospective phase I clinical trial was to analyze safety of two doses of fluorescent rhodamine-labeled cationic liposomes (LDF01) in head and neck squamous cell carcinoma (HNSCC). Patients had resectable UICC stadium I-IV A HNSCCs. LDF01 was administered before tumor resection under general anesthesia as an intravenous infusion with effective lipid doses of 0.5 or 2 mg/kg b.w., respectively. In addition to clinical monitoring for safety assessment, tumor biopsies were taken during the surgical procedure for fluorescence histological analysis. Eight patients were assigned to the two dose groups. During safety follow-up no clinically relevant adverse events occurred. Fluorescence histology revealed some evidence of favorable selectivity of LDF01 for tumor microvessels in the high-dose group. LDF01 is safe applied as infusion at both tested dose levels. Furthermore, LDF01 can be detected in the vicinity of tumor cells and could be assigned to the microvessel target in individual HNSSC cases. Detailed analysis of targeting properties of LDF01 has to be performed in upcoming clinical phase II trials.