As a part of ongoing studies in developing new potent antimicrobial agents, a series of novel 2-(3-pyridyl)-4,5-disubstituted thiazoles was efficiently synthesized and characterized by spectral and elemental analyses. The newly synthesized compounds were evaluated for their in vitro antimicrobial activity against ten bacterial and five fungal human pathogenic strains using the disc diffusion assay. Among the synthesized compounds, 5-acetyl-4-methyl-2-(3-pyridyl)thiazole (5) exhibited twofold antibacterial activity of ampicillin in inhibiting the growth of Staphylococcus epidermidis (MIC 0.24 μg/mL) and also showed equipotent antifungal activity with amphotricin B against Geotricum candidum (MIC 0.48 μg/mL). From structure-activity relationship (SAR) point of view, increasing the size of the substitutions either at position 4 or 5 on the thiazole nucleus decreased the antimicrobial activity.