In this study, we performed the green synthesis of the silver nanoparticles (AgNPs) using the Borago officinalis plant leaves’ extract as an environmentally friendly reducing agent, and studied in vitro antimicrobial and antifungal activities. The study showed a simple, efficient, and rapid approach. The biomolecules found in the plant extract play a crucial role as both stabilizing and reducing agents. Various instrumental methods, viz UV–Vis, FTIR, Raman, XRD, DLS, SEM, HR-TEM, EDX, and AFM were used to identify the produced AgNPs. The surface plasmon spectra of AgNPs are dark brown in color and center at 433 nm. The spherical shape of the synthesized AgNPs was observed and the average particle size was about 22 nm. Synthesized AgNPs have shown antimicrobial activity with an inhibition zone of 8 mm against Escherichia coli bacteria and no inhibition zone against other bacteria, such as Salmonella Typhi, Bacillus subtilis, and Staphylococcus aureus, in the absence of activity, and only Candida albicans inhibits the inhibition zone of 6 mm while Aspergillus flavus is not inhibited.