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Atopic dermatitis (AD) is a chronic inflammatory disease caused by immune dysregulation. Meanwhile, the supernatant of lactic acid bacteria (SL) was recently reported to have anti-inflammatory effects. In addition, HaCaT keratinocytes stimulated by tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) are widely used for studying AD-like responses. In this study, we evaluated the anti-inflammatory effects of SL from lactic acid bacteria (LAB) on TNF-α/IFN-γ-induced HaCaT keratinocytes, and then we investigated the strains' probiotic properties. SL was noncytotoxic and regulated chemokines (macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC)) and cytokines (interleukin (IL)-4, IL-5, IL-25, and IL-33) in TNF-α/IFN-γ-induced HaCaT keratinocytes. SL from Lacticaseibacillus rhamnosus MG4644, Lacticaseibacillus paracasei MG4693, and Lactococcus lactis MG5474 decreased the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). Furthermore, the safety of the three strains was demonstrated via hemolysis, bile salt hydrolase (BSH) activity, and toxicity tests, and the stability was confirmed under simulated gastrointestinal conditions. Therefore, L. rhamnosus MG4644, L. paracasei MG4693, and Lc. lactis MG5474 have potential applications in functional food as they are stable and safe for intestinal epithelial cells and could improve atopic inflammation.