In this study, four novel urea-thiourea hybrids bearing 1,4-naphthoquinone moiety were synthesized by the reaction of 2,3-diaminonaphthalene-1,4-dione and various isothiocyanate derivatives in 75-88% yield, and their molecular structures were characterized by using H-1/C-13 NMR, FT-IR and HRMS techniques. Urea-thiourea hybrids have been known for their anti-inflammatory activities. Therefore, in our study, we aimed to decipher their biological activities on mammalian macrophages. Macrophages were stimulated by LPS and pro-inflammatory TNF, IL6, GMCSF and IL12p40 cytokine production levels were measured in the presence of these derivatives by ELISA. Our results suggest that these derivatives on stimulated mammalian macrophages had anti-inflammatory activities. In order to examine their intracellular mechanism of action, intracellular phosphorylated (activated) PI3K protein levels were measured in the presence of our novel derivatives by flow cytometry. Our results suggest that these novel derivatives had anti-inflammatory activities on mammalian macrophages by blocking the PI3K activation and decreasing the production of pro-inflammatory cytokines. Molecular docking calculations were also employed to elucidate the theoretical binding modes of our studied compounds with PI3K gamma. These derivatives have the potential to be utilized as anti-inflammatory drug agents against versatile inflammatory and autoimmune disorders.