Hippocratea velutina (HV) Afzel's application in wound care suggests its antimicrobial property, although no scientific data confirms its efficacy. Therefore, the antibacterial and antifungal properties of HV leaves are assessed in this study. The methanol extract of HV was evaluated in vitro against a variety of bacterial strains, including common fungal pathogens like Candida albicans and Aspergillus niger, as well as Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and Gram-negative Pseudomonas aeruginosa. The antimicrobial properties were assessed using agar well diffusion assays. The HPLC-UV-DAD-identified compounds of HV were docked against DNA gyrase from Bacillus subtilis and Sterol 14-alpha demethylase from Aspergillus flavus. The findings showed that the extract had strong antibacterial and antifungal properties; the inhibition zones for bacterial and fungal strains ranged from 7.0±0.00 to 20.5±4.95 mm and 10.5 ± 0.71 to 12 ± 0.00 mm, respectively, suggesting a broad-spectrum antimicrobial potential. Additionally, the extract demonstrated fungicidal and bactericidal properties at low doses, suggesting its potential as a therapeutic agent. In HV, phenolic compounds such as vitexin and rutin were detected. Sterol 14-alpha demethylase and DNA gyrase showed persistent interactions with rutin and vitexin. These compounds may be working in concert to provide the antimicrobial effects observed. From this study, Hippocratea velutina may be a valuable substitute for conventional antimicrobial medicines, with possible uses in the medical and pharmaceutical sectors. It is recommended that further studies be conducted to evaluate its safety, effectiveness and potential for commercialization, including in vivo investigations and clinical trials.