<p>Acoustic tweezers are devices that use acoustic waves for contactless particle trapping and manipulation. They provide advantages typical of ultrasound-based techniques, such as minimal thermal effects and high biocompatibility, making them ideal for handling fragile biological samples. By using different transducer configurations and adjusting acoustic parameters, acoustic tweezers can operate on particles across various scales — from nanometres to millimetres — meeting several engineering, biological and medical needs. However, the use of acoustic tweezers in biomedical contexts still requires further optimization to broaden their applications and achieve an impact comparable to that of optical tweezers. This Primer discusses the fundamental principles of acoustic tweezers and outlines their typical experimental set-ups. We showcase advances in applications such as force spectroscopy, single-cell analysis, tissue engineering, organismal studies and in vivo procedures. Additionally, we address reproducibility challenges, suggest data-sharing standards and examine current technological limitations. Our goal is to empower researchers with the foundational knowledge needed to effectively apply acoustic tweezers, fostering their broader adoption in precision biology and medicine.</p>