Leishmaniasis is a neglected tropical disease, and there is an emerging need for the development of effective drugs to treat it. To identify novel compounds with antileishmanial properties, a novel series of functionalized spiro[indoline-3,2′-pyrrolidin]-2-one/spiro[indoline-3,3′-pyrrolizin]-2-one23a–f,24a–f, and25a–gwere prepared from natural-product-inspired pharmaceutically privileged bioactive sub-structures, i.e., isatins20a–h, various substituted chalcones21a–f, and22a–camino acids, via 1,3-dipolar cycloaddition reactions in MeOH at 80 °C using a microwave-assisted approach. Compared to traditional methods, microwave-assisted synthesis produces higher yields and better quality, and it takes less time. We report here the in vitro antileishmanial activity againstLeishmania donovaniand SAR studies. The analogues24a,24e,24f, and25dwere found to be the most active compounds of the series and showed IC50values of 2.43 µM, 0.96 µM, 1.62 µM, and 3.55 µM, respectively, compared to the standard reference drug Amphotericin B (IC50= 0.060 µM). All compounds were assessed for Leishmania DNA topoisomerase type IB inhibition activity using the standard drug Camptothecin, and24a,24e,24f, and25dshowed potential results. In order to further validate the experimental results and gain a deeper understanding of the binding manner of such compounds, molecular docking studies were also performed. The stereochemistry of the novel functionalized spirooxindole derivatives was confirmed by single-crystal X-ray crystallography studies.