Infectious diseases have always been a significant challenge to global public health, and the pandemic caused by the novel coronavirus (SARS-CoV2) has once again sounded the alarm. Bacteria are one of the most important pathogens of infectious diseases. Although antibiotics can effectively control most bacterial infections, the emergence of multi-drug-resistant and pan-drug-resistant strains in recent years may bring humans back to the pre-antibiotic era. One of the most effective strategies to address this daunting challenge is to acquire new antibiotics. Antibiotics with novel targets that target pathogenic virulence factors without causing lethal pressure on bacteria have a low probability of inducing resistance. Therefore, it has the advantages that traditional-killing antibiotics do not have. Due to the long-term interaction with the host, pathogenic bacteria have acquired virulent factors that can effectively escape the immune recognition of the host. Studying these infectious factors can often reveal the pathogen recognition mechanism that is almost impossible to obtain under normal conditions. Given that most virulent factors are proteins with unknown functions, their functional analysis is highly innovative and is expected to reveal breakthrough mechanisms. In this particular Methods Collection, we aim to collect original research relating to current biotechniques in studying virulence factors of microbes. Submissions on new methodologies for drug delivery, such as nanoparticles, liposomes, dendrimers, and solid lipid nanocarriers, that can help combat infectious diseases are welcome.