This research aimed to develop nanosuspensions as carriers of Annona muricata acetogenins (ACGs) using different concentrations of soy lecithin (SL), hydroxypropyl-β-cyclodextrin (βCD) and the dispersion method (low agitation, middle agitation and ultrasound) on particle size and polydispersity index (PDI). A response surface analysis was performed to find an optimized, validated and characterized nanosuspension (OβCDSL-ACGs-NSps) and to measure its antibacterial activity. The best conditions to formulate OβCDSL-ACGs-NSps with spherical nanoparticles (133.77 nm), PDI (0.20) and good stability (−37 mV) were 10 mg/mL SL, 0.16% βCD and ultrasound as a dispersion method. Nanoparticles from OβCDSL-ACGs-NSps exhibited a high entrapment (85.33%) and encapsulation efficiency (56.09%). The highest release (84.15%) of ACGs was observed until 36 h. OβCDSL-ACGs-NSps also showed significant antibacterial inhibition against E. faecalis (87%) and L. monocytogenes (75%) at 320 µg/mL. βCDSL-ACGs-NSps is an attractive and promising alternative to improve the solubility and bioavailability of ACGs in pharmaceutical applications.