Anthracnose, caused by Colletotrichum spp., is one of the major challenges in chili cultivation and is responsible for significant yield losses. This study aimed to develop a nano-emulsion fungicide formulation based on neem oil (Azadirachta indica), citronella oil (Cymbopogon nardus), and liquid smoke for controlling Colletotrichum capsici and Colletotrichum acutatum in vitro. The nano-emulsion was prepared using a high-speed homogenizer to achieve a droplet size below 250 nm. The formulations were characterized using Dynamic Light Scattering (DLS) and High-Resolution Transmission Electron Microscopy (HRTEM) to determine the optimal composition, while the chemical profile of the formulation was analyzed using Gas Chromatography–Mass Spectrometry (GC–MS). Safety assessments included phytotoxicity, ocular irritation, and acute oral and dermal toxicity tests. The optimal formulation, designated as MSA3, produced droplets with an average size of 240.1 nm and a low polydispersity index (PDI) of 0.155. In vitro antifungal assays revealed that at a 2.0% concentration, the nano-formulation (MSA3) was as effective as the commercial fungicide in controlling C. capsici, whereas significant inhibition of C. acutatum was observed only at concentrations above 4.0%. This formulation exhibited no phytotoxicity effects and improved chili seed germination by up to 20%. Oral toxicity assessment classified MSA3 as non-hazardous, while dermal exposure indicated a moderate hazard level. These results indicate that the nano-emulsion fungicide formulated with neem oil, citronella oil, and liquid smoke at this formulation holds considerable potential as an environmentally friendly alternative to conventional synthetic fungicides in sustainable agriculture.