Background <p>Invasive needling procedures in physiotherapy can be performed using palpation or ultrasound guidance. While ultrasound offers greater precision and safety, the influence of healthcare professional experience and specialization in this field on performance has not yet been fully explored. The aim was to compare the accuracy, efficiency, safety, and perceived workload of ultrasound-guided versus palpation-guided needling procedures among physiotherapists with different levels of experience and specialization.</p> Methods <p>Twenty-seven physiotherapists (students, non-specialized and specialized physiotherapists) performed 20 needling tasks each, on cadaveric specimens, targeting the ulnar nerve in the cubital tunnel. Procedures were conducted under two guidance conditions (ultrasound vs. palpation) and two handling modalities (with or without handpiece). Performance metrics (e.g., distance to target, structures affected) and subjective workload were recorded.</p> Results <p>Specialized physiotherapists demonstrated significantly greater precision, efficiency, and safety than the other groups (<i>p</i> &lt; 0.001). Students and non-specialized professionals had similar performance, particularly in terms of precision and control. Subjective workload was lower in specialists, especially under ultrasound guidance.</p> Conclusion <p>Ultrasound guidance improves needling outcomes, especially when performed by trained specialists. General clinical experience without focused ultrasound training may be insufficient to ensure optimal procedural control. These findings support the integration of ultrasound training in advanced physiotherapy programs and the need to seek strategies that enhance learning.</p>