Antimicrobial drugs are among the most commonly used and, unfortunately, misused pharmaceuticals. This broad use has resulted in the emergence of antibiotic-resistant bacteria, producing an increasing need for new treatments. However, the development of novel antimicrobial medications has slowed substantially, with only a few new agents entering clinical practice each year, the majority of which are not particularly revolutionary. This chapter discusses the mechanism of action of antibiotics, as well as some insights into novel potential antibacterial targets and their importance. Understanding the molecular principles underpinning bacterial replication has substantially helped the focused development of compounds aimed to inhibit this process. This chapter explores key existing mechanisms and innovative strategies for achieving effective antimicrobial action. Inhibition of bacterial cell wall synthesis, cell membrane disruptions, inhibition of protein synthesis via targeting different ribosomal units, disruption in nucleic acid metabolism by binding with some important enzymes like RNA polymerase and topoisomerase, inhibition of folate synthesis, and inhibition of non-nucleotide reverse transcriptase enzymes are the mechanisms of conventional antimicrobial action of various antimicrobial agents. Recent antimicrobial research focuses on innovative strategies to combat resistant pathogens with precision. Benzamide-based molecules target Staphylococcus aureus by inhibiting lipoteichoic acid synthesis and FtsZ protein, while FabI and KAS enzyme inhibitors disrupt bacterial fatty acid synthesis, etc. As to harness the impact of nanozyme to combat antimicrobial resistance, the brief mechanisms of nanozymes also be involved in this chapter.