The emergence of resistant bacterial strains mainly due to misuse of antibiotics has seriously affected our ability to treat bacterial illness, and the development of new classes of potent antimicrobial agents is desperately needed. In this study, we report the efficient synthesis of a new pyrazoline-pyridine containing ligand L1 which acts as an NN-donor for the formation of a novel silver (I) complex 2. The free ligand did not show antibacterial activity. High potency was exhibited by the complex against three Gram-negative bacteria, namely Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumanii with the minimum inhibitory concentration (MIC) ranging between 4 and 16μg/mL (4.2-16.7μM), and excellent activity against the fungi Candida albicans and Cryptococcus neoformans (MIC ≤ 0.25μg/mL = 0.26μM). Moreover, no hemolytic activity within the tested concentration range was observed. In addition to the planktonic growth inhibition, the biofilm formation of both Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa was significantly reduced by the complex at MIC concentrations in a dose-dependent manner for Pseudomonas aeruginosa, whereas a biphasic response was obtained for MRSA showing that the sub-MIC doses enhanced biofilm formation before its reduction at higher concentration. Finally, complex 2 exhibited strong DNA binding with a large drop in DNA viscosity indicating the absence of classical intercalation and suggesting the participation of the silver ion in DNA binding which may be related to its antibacterial activity. Taken together, the current results reveal that the pyrazoline-pyridine silver complexes are of high interest as novel antibacterial agents, justifying further in vitro and in vivo investigation.