Due to the high failure rates associated to endodontic disinfection, this study aimed to investigate the antibacterial properties of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with Ca(OH)2 for endodontic disinfection procedures. Ca(OH)2 NPs production and physicochemical characterization were carried out as well as multiple antibacterial tests using three bacterial strains and an ex vivo model of endodontic infection with extracted human teeth. Agar diffusion test and broth dilution determined the inhibition growth zones (n = 5) and the minimal inhibitory concentration (MIC, n = 5), respectively. Cell viability was assessed using Live/Dead staining with confocal microscopy (n = 5). Data was analysed using ANOVA followed by post-hoc analysis. After 24 h of incubation, Ca(OH)₂ NPs demonstrated a MIC of 10 µg/mL for Porphyromonas gingivalis (p < 0.001) and Enterococcus faecalis and 5 µg/mL for Fusobacterium nucleatum (p < 0.001). Although the agar diffusion test did not exhibit any inhibition area for Ca(OH)2 nor for Ca(OH)₂ NPs, this was probably due to the buffering effect of the agar medium. However, the antibacterial capacity was confirmed in an ex vivo model, where instrumentalized teeth were infected with Enterococcus Faecalis and treated after 28 days of culture. A significant reduction in bacterial metabolic activity was confirmed for Ca(OH)2 NPs (40 % reduction with a single dose) and confirmed by Live/Dead staining. In conclusion, Ca(OH)₂-loaded PLGA NPs present promising antibacterial efficacy for endodontic disinfection procedures.