Significance: Venous leg ulcers (VLUs) are susceptible to microbial invasion, and serious complications can result without the timely control of infection. Diagnosis of wound infection is primarily based on subjective clinical characteristics and patient-reported symptoms, and the treatment with antimicrobials has not consistently shown improvement in healing outcomes. This is a review of studies using bacterial cultures and/or new molecular-based methods associating microbial bioburden with healing outcomes in VLU patients, with the goal of guiding future studies to better determine significant patterns of microbial involvement in chronic wounds. Recent Advances: Studies reviewed here use cultivation-based identification of bacteria and next-generation sequencing of the bacterial 16S rRNA gene to gain insight into microbial bioburden in VLUs. Further application of sophisticated DNA sequencing and bioinformatic analyses has the potential to revolutionize our ability to further discern, with high resolution, complex microbial communities in chronic wounds. Critical Issues: Few previous studies of microbial bioburden in VLUs have incorporated the knowledge of clinical treatments, which includes close monitoring of patients' symptoms and responses to therapy. Thus, wound care practitioners are currently without evidence-based guidance for the diagnosis and treatment of wound infections. Future Directions: Clinically relevant breakthroughs are possible by combining advanced microbial detection techniques with improved study designs that reflect clinical practices. Well-designed longitudinal studies have great potential to lead to better evidence-based diagnosis of chronic wounds. A greater understanding of microbial bioburden in chronic wounds is likely to lead to better therapies that speed healing and prevent wound infection without risking the development of antimicrobial resistance.