Cross-linked polysiloxane microspheres containing a large number of SiOH groups were modified by introduction of organic thiol groups, which were further used for the functionalization of the microspheres with silver thiolate groups. The microspheres were characterized by Si-29 MAS NMR, C-13 MAS NMR, SEM, XPS and elemental analysis. They were tested as biocides against selected Gram-positive and Gram-negative bacteria strains and exhibited high bactericidal activity. Separately, linear polysiloxane polymers equipped with organothiol groups and loaded with silver were synthesized. Their antibacterial activity was compared with that of silver thiolate-functionalized microspheres. Different shape of particles and a different form of silver explained somewhat lower activity of polymers.