Rhodomyrtus tomentosa, a flowering plant of Myrtaceae family from southern and southeastern Asia, was known to possess a rich source of structurally diverse and various biological activities. In this study, the inhibitory effect of R. tomentosa fruit extract (RFE) on allergic responses in calcium ionophore A23187-activated RBL-2H3 mast cells was investigated. The result showed that RFE was able to inhibit mast cell degranulation via decreasing β-hexosaminidase release and intracellular Ca2+ elevation at the concentration of 400 μg/ml. Moreover, the suppressive effects of RFE on the production of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were evidenced. In addition, RFE effectively scavenged DPPH radical and suppressed the reactive oxygen species generation in a dose-dependent manner. Notably, the pretreatment of RFE caused the downregulation of tyrosine kinase Fyn phospholipid enzyme phospholipase Cγ (PLCγ), extracellular-signal-regulated kinase (ERK), and nuclear factor kappa B (NF-κB) phosphorylation. These results indicated that RFE could be a promising inhibitor of allergic responses and may be developed as bioactive ingredient for prevention or treatment of allergic diseases.