The different analogues of ebselen—unsubstituted benzisoselenazol-3(2H)-one (2a) 2-pyridylbenzisoselenazol-3(2H)-ones (2b–h) and 7-azabenzisoselenazol-3(2H)-ones (3a–j) were designed as new selenium-containing antiviral and antimicrobial agents and synthesized. Some of them were found in the antiviral assay in vitro to be strong inhibitors of cythopatic activity of herpes simplex virus type 1—HSV-1 (compounds 2a,b,f,h, 3a–j) and encephalomyocarditis virus—EMCV (compounds 2a,h, 3a–f,k,l). The compounds 2a,h and 3a–e,j were found to have an appreciable activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus) in vitro, some of them inhibited growth of pathogenic yeasts (Candida albicans) (3a,b) and filamentous fungi (3a–e,f).