As natural products, non-ribosomal peptides (NRPs) exhibit biological activities with a broad spectrum, including anticancer, antibiotic and immunosuppression. Among U.S. Food and Drug Administration (FDA) approved drugs, fungal NRPs are a major category of pioneering pharmacological agents like immunosuppressive cyclosporine, antibacterial cephalosporin and antifungal echinocandins. Under the catalysis of complicated multimodular enzyme complexes known as non-ribosomal peptide synthetases (NRPSs), NRPs are synthesized with three core domains: adenylation (A), thiolation domain/peptidyl carrier protein (T/PCP) and condensation (C), which collectively form repetitive modules responsible for activating and incorporating specific amino acids or hydroxycarboxylic acid building blocks into the growing peptide chains. Beyond the core domains, optional domains are exemplified by epimerization (E), heterocyclization (Cy) and oxidation (Ox), facilitating the customization of the building blocks. These domains and the variability in the number of modules with NRPs significantly contribute to the structural diversity of the skeletons. Furthermore, post-modifications to the structural skeletons yield potent pharmacological groups for NRPs, contributing significantly to their structural diversity and biological activities, which not only provide opportunities for discovering naturally sourced and active NRPs, but also opens avenues for modifications to create non-natural NRPs via synthetic biological technology. To date, numerous strategies have been employed for developing NRPs, including heterologous expression, transcriptional factor activation, precursor-directed biosynthesis, mutasynthesis, combinatorial biosynthesis and enzyme engineering. This review summarizes the progress in research on fungal NRPs, encompassing their bioactivities, biosynthetic pathways, enzymatic reaction mechanisms and metabolic engineering. A comprehensive understanding of fungal NRPs biosynthesis not only benefits for deciphering the corresponding enzymatic assembly mechanism, but also serves as a guidance for advancing novel fungal NRPs and their derivatives, thereby paving the way for developing potential drug candidates from NRPs.