Nanotechnology has focused heavily on using biological components instead of toxic chemicals to synthesise and stabilise nanoparticles. Bioactive compounds obtained from fungi and yeast are particularly effective for this purpose. These microorganisms can be readily cultivated and sustained in a lab environment due to their rapid growth and ability to produce extracellular enzymes. By employing these reducing enzymes, fungi, and yeast can generate metal nanoparticles and nanostructures either intracellularly or extracellularly. Fungal nanoparticles are increasingly recognised as valuable in therapeutic and biomedical applications due to their unique properties and eco-friendly production methods. This chapter presents an in-depth overview of the green synthesis of nanoparticles, emphasising fungal-mediated synthesis. It covers both intracellular and extracellular synthesis, highlighting the benefits and potential uses of each method. This chapter also examines the various biomedical applications of nanoparticles, including their use in drug delivery, wound healing, and disease detection and diagnosis. It specifically focuses on the role of nanoparticles in immunohistochemistry, proteomics, nano-DNA technology, biosensing, and medical imaging. Additionally, the therapeutic potential of nanoparticles is explored, particularly in the treatment of diseases like type 1 diabetes, Alzheimer’s, HIV/AIDS, and cancer treatments such as gene therapy, immunotherapy, and radiation therapy.