Vernonia greggii belongs to the Asteraceae family, and some members of this family have been reported to possess anticancer properties. This study evaluated the antiproliferative effect of V. greggii methanol extract (ME), both in its free form and encapsulated into poly(lactic-co-glycolide) (PLGA) nanoparticles (NPs), on human cervical cancer cells (HeLa) and human epidermal keratinocytes (HaCaT). The extract was subsequently sub-fractionated into n-hexane (F-He), methanol (F-Me), and distilled water (F-Ac) fractions, and their antiproliferative effects were assessed. Time-dependent toxicity on HeLa cells was observed for the free-form fractions, with the F-Me fraction showing the highest efficacy compared to the others. Additionally, an NP formulation based on PLGA and F-Me (NPs F-Me) was developed, achieving 64.21% encapsulation efficiency and 11.38% drug loading. The NPs had an average size of 146.9 nm, a polydispersity index (PDI) of 0.103, and a ζ-potential of 23.3 mV. NPs F-Me were tested on HeLa and HaCaT cells, with toxicity observed at concentrations of 300 and 500 μg/mL, affecting tumor cell morphology. Furthermore, the hemolytic activity of F-Me and NPs F-Me was evaluated. The major bioactive compounds in the F-Me fraction were identified using Liquid Chromatography–Mass Spectrometry (LC-MS). These findings suggest that the F-Me fraction of V. greggii exerts an antineoplastic effect both in its free form and when encapsulated in nanoparticles.