Antibiotics are frequently used to treat, prevent, or control bacterial infections, but in recent years, infections resistant to all known classes of conventional antibiotics have significantly grown. The development of novel, nontoxic, and nonincursive antimicrobial methods that work more quickly and efficiently than the present antibiotics is required to combat this growing public health issue. Here, Co(II) and Zn(II) derivatives of tetrakis(1-methylpyridinium-4yl)porphyrin [H 2 TMPyP] 4+ as a tetra(ρtoluenesulfonate) were synthesized and purified to investigate their interactions with DNA (pH 7.40, 25 °C) using UV−vis, fluorescence techniques, and antimicrobial activity. UV−vis results showed that [H 2 TMPyP] 4+ had a high hypochromicity (∼64%) and a substantial bathochromic shift (Δλ, 14 nm), while [Co(II)TMPyP] 4+ and [Zn(II)TMPyP] 4+ showed little hypochromicity (∼37%) and a small bathochromic shift (Δλ, 3−6 nm). Results reveal that [H 2 TMPyP] 4+ interacts with DNA via intercalation, while Co(II)-and [Zn(II)TMPyP] 4+ interact with DNA via outside self-stacking. Fluorescence results also confirmed the interaction of [H 2 TMPyP] 4+ and the metalloporphyrins with DNA. Results of the antimicrobial activity assay revealed that the metalloporphyrins showed inhibitory effects on Gram-positive and Gram-negative bacteria and fungi, but that neither the counterions nor [H 2 TMPyP] 4+ exhibited any inhibitory effects. Mechanism of antimicrobial activities of metalloporphyrins are discussed.