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Abstract Eleven new and three reported half-sandwich Ru(II) arene complexes were synthesized using [Ru(η6-benzene)Cl(μ-Cl)]2 and [Ru(η6-toluene)Cl(μ-Cl)]2 and four pyridine carboxylic acid-based ligands (dicarboxylic acids and halogen derivatives). The structures and purity of synthesized compounds were confirmed using 1H and 13C NMR spectroscopy, infrared spectroscopy, mass spectrometry, and elemental analysis. The stability of synthesized compounds in dimethyl sulfoxide solution was confirmed using 1H NMR spectroscopy. The seven ligands, two complex precursors (CP1 and CP2), and 14 half-sandwich Ru(II) picolinate complexes (C1–C14) were evaluated for in vitro antibacterial and antifungal activity against pathogens, such as Staphylococcus aureus, Bacillus cereus, Proteus mirabilis, Klebsiella pneumoniae, Ecsherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis, Enterobacter aerogenes, and yeast Candida albicans, using the microwell-dilution method. Among the tested samples, the ligands showed better inhibitory effect against Gram-positive bacteria when compared to the metal complexes. The most susceptible Gram-negative bacteria was Ecsherichia coli, with a MIC value of 1.25 mg/mL, for C3, C6, and C10. All synthesized complexes showed similar, slightly better activity against Candida albicans. GRAPHICAL ABSTRACT