Background Cnidarian venoms and extracts have shown a broad variety of biological activities including cytotoxic, antibacterial and antitumoral effects. Most of these studied extracts were obtained from sea anemones or jellyfish. The present study aimed to determine the toxic activity and assess the antitumor and antiparasitic potential of Palythoa caribaeorum venom by evaluating its in vitro toxicity on several models including human tumor cell lines and against the parasite Giardia intestinalis. Methods The presence of cytolysins and vasoconstrictor activity of P. caribaeorum venom were determined by hemolysis, PLA2 and isolated rat aortic ring assays, respectively. The cytotoxic effect was tested on HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). An in vivo toxicity assay was performed with crickets and the antiparasitic assay was performed against G. intestinalis at 24 h of incubation. Results P. caribaeorum venom produced hemolytic and PLA2 activity and showed specific cytotoxicity against U251 and SKLU-1 cell lines, with approximately 50% growing inhibition. The venom was toxic to insects and showed activity against G. intestinalis in a dose-dependent manner by possibly altering its membrane osmotic equilibrium. Conclusion These results suggest that P. caribaeorum venom contains compounds with potential therapeutic value against microorganisms and cancer.