Magnesium oxide nanoparticles (MgO NPs) have emerged as potential materials for various biomedical applications due to their unique physicochemical properties, including biodegradability, biocompatibility, cationic capacity, high stability and redox properties. MgO NPs have become an attractive platform to combat microbes and may be a promising alternative to overcome challenges associated with eliminating microbial biofilms and antibiotic resistance. Hence, due to the increasing use of MgO NPs in biomedicine, new synthetic strategies for MgO NPs are necessary. MgO NPs synthesised using green methods are non-toxic, eco-friendly and have high stability for a wide range of biological, medical and catalytic applications. This review presents the recent advances in biosynthesis strategies of MgO NPs by diverse bio-templates, such as plant, bacterial, fungal and algal extracts. Its photocatalytic properties show a suitable inhibitory function against pathogenic agents, such as microbial proliferation, biofilm formation and fungal growth. Furthermore, MgO NPs and relevant nanocomposites are comprehensively discussed regarding the mechanisms of their effect on microbes, biofilms and fungal strains, as well as challenges and future perspectives.