A simple hydrothermal route is employed to synthesize pure copper indium disulfide (CIS) and CIS nanoparticles (NPs) mediated by various natural plant extracts. The plant extracts used to mediate are (neem), (basil), (coconut), (aloe), and (turmeric). The tetragonal unit cell structure of as-synthesized NPs is confirmed by X-ray diffraction. The analysis by energy-dispersive X-rays shows that all the samples are near-stoichiometric. The morphologies of the NPs are confirmed by high-resolution scanning and transmission modes of electron microscopy. The thermal stability of the synthesized NPs is determined by thermogravimetric analysis. The optical energy band gap is determined from the absorption spectra using Tauc's equation. The antimicrobial activity analysis and the estimation of the minimum inhibitory concentration (MIC) value of the samples are performed for , , , and pathogens. It shows that the aloe-mediated CIS NPs possess a broad inhibitory spectrum. The best inhibitory effect is observed against whereas the least effect was exhibited against . The least MIC value is found for aloe-mediated CIS NPs (0.300 mg/mL) against , and , along with basil-mediated NPs against . The antioxidant activity study showed that the IC value to inhibit the scavenging activity is maximum for the control (vitamin C) and minimum for pure CIS NPs. The in vivo cytotoxicity study using brine shrimp eggs shows that the pure CIS NPs are more lethal to brine shrimp than the natural extract-mediated CIS NPs. The in vitro cytotoxicity study using the human lung carcinoma cell line (A549) shows that the IC value of turmeric extract-mediated CIS NPs is minimum (15.62 ± 1.58 μg/mL). This observation reveals that turmeric extract-mediated CIS NPs are the most potent in terms of cytotoxicity toward the A549 cell line.