A new synthetic approach to the 1,2,5-oxadiazine ring system is described. 2-Substituted or 2,4-disubstituted 2H-1,2,5-oxadiazine-3,6(4H,5H)-dione derivatives 4 were prepared by cyclisation of hydroxamic acids 3 derived from N-(1-benzotriazolylcarbonyl)-amino acids 1. The structures of the synthesised compounds were fully characterised by IR, 1H and 13C NMR spectroscopy and elemental analysis. The aim of this study was to evaluate biological activity of the newly synthesised oxadiazine derivatives. Cytotoxic and cytostatic activities were tested on two cell lines (HeLa and GMK) and evaluated by MTT-test. Two human DNA viruses (adenovirus 7 and herpesvirus 1) and two human RNA viruses (coxsackievirus B5 and echovirus 7) were used in the antiviral test. Selected biological studies indicated that 2-phenyl- -2H-1,2,5-oxadiazine-3,6(4H,5H)-dione (4a) and 4-benzyl-2-phenyl-2H-1,2,5-oxadiazine-3,6(4H,5H)-dione (4c) statistically significantly inhibited cell growth. A minor antiviral effect was observed upon adenovirus, herpesvirus and enteroviruses.