Nanoparticles (1 to 100 nm) have unique physical and chemical properties, which makes them suitable for application in a vast range of scientific and technological fields. In particular, metal nanoparticle (MNPs) research has been showing promising antimicrobial activities, paving the way for new applications. However, despite some research into their antimicrobial potential, the antimicrobial mechanisms are still not well determined. Nanoparticles’ biosynthesis, using plant extracts or microorganisms, has shown promising results as green alternatives to chemical synthesis; however, the knowledge regarding the mechanisms behind it is neither abundant nor consensual. In this review, findings from studies on the antimicrobial and biosynthesis mechanisms of MNPs were compiled and evidence-based mechanisms proposed. The first revealed the importance of enzymatic disturbance by internalized metal ions, while the second illustrated the role of reducing and negatively charged molecules. Additionally, the main results from recent studies (2018–2022) on the biosynthesis of MNPs using microorganisms were summarized and analyzed, evidencing a prevalence of research on silver nanoparticles synthesized using bacteria aiming toward testing their antimicrobial potential. Finally, a synopsis of studies on MNPs applied to cultural heritage materials showed potential for their future use in preservation.