Leishmaniasis is a neglected zoonotic disease, for which no vaccine is available. The existing drug treatments are associated with significant side effects, including hepatic, renal, and cardiac complications. In this study, we evaluated the toxicity and safety profiles of two formulations: NanoPluNO, comprising oleic acid, Pluronic-127, and a ruthenium complex ([Ru(bpy)2(NO)(SO3)](PF6)); and another one, NanoTwNO, using Tween 80 instead of Pluronic F-127. When tested at concentrations of up to 100 μg/mL, they did not induce cytotoxicity in macrophages, except for NanoPluNO (at 100 μg/mL, with 20% reduction in viability). Acute in vivo toxicity tests (OECD 423) were subsequently conducted on mice, administering doses up to 300 mg/kg — equivalent to 1000 times the effective in vivo leishmanicidal dose. No apparent clinical or laboratory signs of toxicity were observed over a 14-day period. We also performed a subchronic toxicity evaluation (OECD 407), wherein animals were treated daily for 28 days with NanoPluNO and NanoTwNO at a dose of 6 mg/kg per day, equivalent to 20 times the maximum effective in vivo dose against Leishmania. No deaths or significant changes in hematological parameters or serum biochemistry were observed in the NanoTwNO group. However, in the NanoPluNO group, there were two fatalities, as well as two deaths in the Pluronic-127 vehicle control. Moderate hepatic inflammation, along with hepatic and renal vacuolar degeneration, was observed in the NanoPluNO-treated animals. In additional experiments, these nanoemulsions were administered intravenously, and cardiovascular and respiratory functions were assessed through left ventricular pressure-volume loop analysis and electrocardiography. These formulations were infused over 1 hour at rates of 0.3 μg/kg/min, 3.0 μg/kg/min, 10 μg/kg/min, and 30 μg/kg/min. Following the final infusion rate (30 μg/kg/min), corresponding to a cumulative dose of 1.8 mg (6 times the maximum effective dose), a reduction in blood pressure (average decrease of 16 mmHg) and an increase in cardiac output (from 40 to 45 mL/min) were observed. No significant changes were detected in electrocardiographic parameters or respiratory rate or amplitude. NanoTwNO formulation demonstrated low toxicity and a favorable safety margin, making it the preferred candidate for subsequent trials in naturally infected dogs diagnosed with visceral leishmaniasis.