The direct application of heavy metal- and quaternary ammonium-based antibacterial agents can cause inconvenience such as irritation, short-term applicability, discoloration of the tissue, and environmental concerns. The immobilization of these agents on montmorillonite (Mnt) was expected to diminish these effects by hindering direct contact of the ions with the target tissues. The objective of the present study was, therefore, to prepare inorgano(I)- and organo(O)-montmorillonites (I/O-Mnt) and to determine their potential uses in such biomedical applications. Na-montmorillonite (Mnt-Na) was modified by hydrothermal and microwave irradiation methods using Cu2+/Zn2+, and quaternary ammonium and/or anionic surfactants. The effect of the structures formed by immobilization on Mnt surfaces on antibacterial activity was investigated. Quaternary ammonium surfactants were cetyltrimethyl ammonium bromide (CTAB) with a linear alkyl chain, cetylpyridinium chloride (CPC) with a single aromatic ring, and benzethonium chloride (BZT) with double aromatic rings. N-lauroyl sarcosinate (SR) was the anionic surfactant. The samples were subjected to thermogravimetric (TGA) and scanning electron microscopy (SEM) analyses. Desorption tests showed that the antibacterial efficacy against Streptococcus mutans stemmed from I/O-Mnt and not from the ions released from the material surfaces to the aqueous phase. The results of the antibacterial studies showed that the existence of a linear alkyl chain and a double aromatic ring were the structural factors causing the greatest antibacterial effect. The time-kill tests revealed that Mnt-CTA, Mnt-BZT, and Mnt-CP-SR were effective against Streptococcus mutans within 5 min of contact. With the new findings, they were identified as possible selective and potent bactericidal agents and promising candidates for biomedical applications.