Copper is an essential trace redox active metal and its concentration in human body is increased during infections. Given the potential for copper ions to modulate antibiotic activity, this study investigates the interactions between Cu2+ and levofloxacin (LVX) in vitro, under pH conditions representative of S. pneumoniae-induced pneumonia, with subsequent assessment of the resulting antibacterial and cytotoxic effects. Complex formation between Cu2+ and LVX is observed at both, physiological (pH 7.4), as well as in acidic conditions (pH 4) associated with the metabolic activity of S. pneumoniae. At both investigated pHs monoligand and bisligand Cu2+ complexes of LVX were detected by UV-Vis, EPR and cyclic voltammetry. Their abundance depends on pH and molar ratio of LVX and Cu2+. The physiological pH 7.4 and higher LVX/Cu2+ molar ratios shift equilibrium toward formation of bisligand complex. In both complexes Cu2+ coordinates with LVX through the carboxylic and β-keto oxygen atoms. The effect of Cu2+ coordination with LVX on its antibacterial and cytotoxic activity were investigated. Formation of Cu2+-LVX complexes enhance antibacterial activity of LVX against S. pneumoniae, E. coli, and K. pneumoniae at both pH values, but reduce activity against S. aureus compared to LVX alone. Additionally, Cu2+-LVX complexes exhibit higher cytotoxicity toward normal lung fibroblasts than A549 carcinoma cells with a more pronounced effect under acidic conditions. In vitro evaluation of antibacterial and cytotoxic activity of LVX in the presence of Cu2+ ions highlights the Janus-faced behavior of the Cu2+-LVX complexes, characterized by simultaneous beneficial improvement of antimicrobial activity and harmful cytotoxicity to lung fibroblasts.