Background: Inspired by natural anti-inflammatory quaternary benzo[C]phenanthridine alkaloids, novel 2-phenylphthalazin-2-ium bromides were previously designed and synthesized. Objective: The anti-inflammatory effect of 2-phenylphthalazin-2-ium bromides was evaluated based on inflammatory cytokines, and their possible mechanism was explored through the NF-κB, TLR4 and MAPK signaling pathways. Methods: The tested concentrations of two compounds were assessed using MTT assay in vitro. Griess assay was used to determine the changes in nitric oxide (NO) in the cell culture supernatant. qRT‒PCR was used to detect the mRNA levels of inflammatory cytokines, such as IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS. The secretion levels of TNF-α and IL-1β were detected by ELISA. Western blot test was used to detect the protein expression of IL-6, IL-10, TLR4, iNOS, NF-κB, p-P38/P38, p- ERK/ERK and p-JNK/JNK. Results: 2-(3,5-Dichlorophenyl)phthalazin-2-ium bromide (2) with a concentration below 1 μg/mL showed no significant effect on the growth inhibition of RAW264.7 cells, so the concentrations of compound 2 used for experiments were set to 0, 0.25, 0.5 and 1 μg/mL. Compared with the blank control group, the model group showed increased release of NO, transcription levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS (p< 0.05), and ratios of p-P38/P38, p-ERK/ERK, p-JNK/JNK (p< 0.05). Compared with the model group, the sample groups displayed decreased NO release and reduced transcriptional levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4, and iNOS and reduced protein expression ratios of IL-6, IL-1ß, IL-10, TNF-α, NF-κB, TLR4, iNOS, p-P38/P38, p-ERK/ERK and p- JNK/JNK (p< 0.05). Conclusion: This study showed that 2-phenylphthalazin-2-ium bromides partially protected macrophages from the LPS-induced inflammatory response by suppressing TLR4-NF-κB/MAPK signaling and reducing NO production.