This study was carried out to synthesize and characterize vanadium nanoparticles (VNPs) using orange peel extract and to evaluate the antimicrobial, antioxidant, and anticancer activities of the biosynthesized VNPs. The antifungal activity was tested against Fusarium graminearum, Alternaria alternata, Candida albicans, Aspergillus niger, and Aspergillus flavus. However, the antibacterial activity was tested against Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and Gram-negative bacteria (Yersinia enterocolitica, Salmonella typhimurium, and Escherichia coli). The antioxidant activity was determined using the DPPH assay, and the anticancer activity was tested against the MCF-7 breast cell line. The biosynthesized VNPs were mostly square with an average size of 80 ± 3 nm and zeta potential of − 50 ± 6 mV. VNPs showed strong antifungal and antibacterial activities at a concentration of 100 µg/mL against the tested fungi and bacteria. These biosynthesized VNPs showed strong antioxidant reaching 91.65% at a concentration of 2000 µg/mL. Moreover, the anticancer activity against MCF-7 breast cell line with IC50 reached 15.99 µg. Therefore, it could be concluded that VNPs with strong antimicrobial, antioxidant, and anticancer activities can be synthesized successfully using orange peel extract and can be used in pharmaceutical and medical applications.