We analyzed, for the first time, the major components and biological properties of the venom of Vespa bicolor, a wasp from South China. Using HPLC and SDS-PAGE, combined with LC–MS/MS, MALDI-TOF-MS, and NMR data to analyze V. bicolor venom (VBV), we found that VBV contains three proteins (hyaluronidase A, phospholipase A1 (two isoforms), and antigen 5 protein) with allergenic activity, two unreported proteins (proteins 5 and 6), and two active substances with large quantities (mastoparan-like peptide 12a (Vb-MLP 12a), and 5-hydroxytryptamine (5-HT)). In addition, the antimicrobial activity of VBV was determined, and results showed that it had a significant effect against anaerobic bacteria. The minimum inhibitory concentration and minimum bactericidal concentration for Propionibacterium acnes were 12.5 µg/mL. Unsurprisingly, VBV had strong antioxidant activity because of the abundance of 5-HT. Contrary to other Vespa venom, VBV showed significant anti-inflammatory activity, even at low concentrations (1 µg/mL), and we found that Vb-MLP 12a showed pro-inflammatory activity by promoting the proliferation of RAW 264.7 cells. Cytotoxicity studies showed that VBV had similar antiproliferative effects against all tested tumor cell lines (HepG2, Hela, MCF-7, A549, and SASJ-1), with HepG2 being the most susceptible. Overall, this study on VBV has high clinical importance and promotes the development of Vespa bicolor resources.