: Currently there are very few effective oral antileishmanial treatments. In this study we evaluated a new bis(pyridine-2-carboxamidine) antileishmanial drug (JNII40_base) and its hydrochloride salt (JNII40_HCl). : The characterization studies performed allowed us to determine the crystallinity, hydration water, and presence of hydrogen bonds in these drugs. Different dissolution methods were employed to predict intestinal absorption. A high-performance liquid chromatography-mass spectrophotometry (HPLC-MS/MS) method was developed for the determination of JNII40 in plasma. : Pharmacokinetic studies in rats of JNII40_base at 100 and 20 mg/kg, and JNII40_HCl at 20 mg/kg, showed a non-linear pharmacokinetic at high doses. An innovative biorelevant medium of phosphate buffer pH 6.8 with polysorbate 80 at 0.6% (/) showed high concentration values for JNII40_base at 30 min, which predicts good intestinal absorption. These results were consistent with the bioavailability data, which exhibited a significant ( < 0.05) increase in maximum plasma concentration (C) and a slight delay in time to maximum (T) compared to JNII40_HCl. Furthermore, the sustained release of JNII40_base in this biorelevant media was related to high plasma concentration values at 24 h (C) observed in bioavailability studies. These plasma concentrations of JNII40_base were above the half-maximal inhibitory concentration (IC) against promastigote and amastigote forms of , which is indicative of effectiveness and should reduce the occurrence of drug resistance during treatments. : The bioavailability and pharmacokinetic data support the consideration of this drug for further in vivo studies as an oral antileishmanial treatment.