Objective: To evaluate antimicrobial and antioxidant properties of saline extract from Tithonia diversifolia leaves by phytochemical bioprospecting, and investigate its safety against animal cells. Methods: The saline extract was prepared, with NaCl (0.15 M), by constant stirring of the dried and pulverized leaves, followed by volume reduction by lyophilization. The extract was phytochemical characterized using ultra-performance liquid chromatography, and total phenol and flavonoid analysis also was performed. The antioxidant capacity was determined through DPPH· radical, the antimicrobial property was evaluated against standard bacteria and fungi, and the viability assays were performed against mice splenocytes. Results: Fifteen compounds were identified belonging to two main classes terpenoids and phenolics. The extract showed 22.185 mg GAE/g of total phenolic compounds and 3.220 mg QE/g of flavonoid. Moreover, extract showed higher antioxidant ability similar to butylated hydroxytoluene a standard molecule [(3.042±0.019) mg AAE/g and (4.12±0.10) mg AAE/g to saline extract and butylated hydroxytoluene, respectively]. The antimicrobial assays demonstrated that the extract had a significant antifungal potential against Candida species and could be used with safety against mice splenocytes, in concentrations lower than 50 μg/mL, promoting higher proliferation in these cells. Conclusions: Saline extract from Tithonia diversifolia leaves presents potential antioxidant, antifungal properties and induces immunostimulation in mice splenocytes.