This study investigates the antifungal potential of encapsulated essential oil (EO) from Baccharis dracunculifolia and nerolidol (NE) within Pluronic® F-127 nanoparticles (NPs). The EO, containing nerolidol, β-caryophyllene, and α-pinene as major bioactive compounds, exhibited superior antifungal activity compared to NE. The NP-EO formulations demonstrated high efficacy against Botrytis cinerea, with inhibition rates ranging from 29.73% to 87.60% and moderate efficacy against Rhizopus sp., with inhibition rates from 11.81% to 32.73%. In comparison, NP-NE showed lower antifungal activity. Both formulations effectively inhibited spore germination, with NP-EO showing greater inhibition compared to NP-NE. The encapsulation efficiency was significantly higher for NP-EO (80.1%) as compared to NP-NE (51.1%), attributed to the complex composition of EO facilitating better encapsulation and retention. Stability studies indicated that both NP formulations were stable at 25 °C for at least 15 days and exhibited changes in particle size and the formation of smaller particle populations at other temperatures (4 °C and 37 °C). Hemolytic activity was low across all NPs, suggesting their safety for food applications. The findings underscore the efficacy and applicability of EO-encapsulated NPs in extending food shelf life and maintaining product quality. The controlled and prolonged release of active compounds, coupled with their antifungal activity and safety, suggests that these NPs represent a promising and innovative approach for food preservation and active packaging development.