Parkinson's disease (PD) is characterized by progressive motor deficits and dopaminergic neuronal loss. Aberrant neuroinflammation contributes to neurodegeneration. Prompted by published case reports describing motor improvement following levofloxacin (LVFX) administration in patients with parkinsonian syndromes, as well as an additional clinical observation from our center, we investigated its effects in cellular and mouse models. In LPS-stimulated BV2 microglial cells, levofloxacin was associated with reduced activity of the TLR4/NF-κB/NLRP3 axis, reducing pro-inflammatory cytokine release and oxidative stress. TLR4 knockdown experiments suggested that TLR4 is involved in mediating this anti-inflammatory action. Conditioned medium from levofloxacin-treated microglia attenuated LPS-induced neuronal viability loss and apoptosis in SH-SY5Y cells. Furthermore, levofloxacin attenuated activation of the TLR4/NF-κB/NLRP3 pathway and glial activation in an LPS-induced mouse model of neuroinflammation. In an MPTP-induced mouse model of PD, levofloxacin alleviated dopaminergic neuronal degeneration and motor deficits. These findings are consistent with clinical observations and preclinical evidence and suggest that levofloxacin may have beneficial effects in models of PD-like pathology, at least in part, by modulating the neuroinflammatory TLR4/NF-κB/NLRP3 axis.