The presence of bioactive peptides in animal venoms has been targeted in scientific research for assessing biological activities, as well as mechanisms of action. A recent study by our group observed hypotensive action of TistH (Tityus stigmurus Hypotensin), a peptide deduced from the transcriptome of T. stigmurus venom gland. The present study aims to analyze TistH structure properties and to evaluate its toxicity on normal and tumor cells, its in vitro antimicrobial activity, as well as its inflammatory effect. Circular dichroism analyses of TistH showed a general predominance of α-helix conformation in TFE (20–70%) and structural stability to pH variations. TistH was not cytotoxic to normal cell lines (3T3, RAW and HEK), and also not to cancer cell lines (HeLa, B16, 786–0, SiHa and HepG2). The peptide did not present inflammatory activity up to 6 h after administered subcutaneously to Swiss mice. It was observed that concentrations of 4–1024 μg/mL of TistH produced no inhibition against the bacteria Staphylococcus aureus, Staphylococcus epidermidis e Pseudomonas aeruginosa. The results of antifungal assays showed a moderate activity of TistH against Candida albicans strain LM-108 and the filamentous fungus Trichophyton rubrum LM-640, with growth inhibition at a concentration of 1024 μg/mL. In contrast, the peptide presented a greater activity (MIC 128 μg/mL) against C. albicans LM-106, Candida tropicalis ATCC 13308 and Aspergillus flavus strains LM-247 and LM-26, fungi that cause oral and vaginal infections, candidiasis and respiratory allergies, respectively. The present data contribute to a better understanding of TistH and its possible use as a bioactive compound. This multifunctional peptide is capable of acting as anti-hypertensive, as well as to inhibit the growth of fungal strains, having low toxicity, which suggests its safety for using as a pharmacological agent.