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Excessive inflammatory response and oxidative stress are the leading causes of periodontal disease (PD). Previously, we reported that Lactobacillus paracasei subsp. paracasei NTU 101-fermented skim milk ethanol extract (NTU101FSMEE) may be useful for developing anti-periodontitis products. In this study, the bioactive ingredient in NTU101FSMEE was isolated and identified as a mixture of tyrosine and lactic acid in 3:1 ratio (3T1L). Treatment of RAW 264.7 and HGF-1 cells with 3T1L significantly reduced the levels of various cytokines participating in the inflammatory response of PD. 3T1L inhibited osteoclastogenesis by decreasing the tartrate-resistant acid phosphatase-related index and suppressed bone resorption by regulating matrix metalloproteinase‐9 and tissue inhibitor of metalloproteinase‐1 levels. Furthermore, 3T1L modulated the intracellular oxidative stress and expression of mitogen-activated protein kinases and the nuclear factor-κB signaling pathway observed in response to lipopolysaccharide. Our findings suggest the potential of 3T1L derived from NTU101FSMEE for anti-periodontitis application.