In this manuscript, we describe the effectiveness of crystal proteins from Gram-positive sporulating bacteria against Acanthamoeba castellanii trophozoites. Light and scanning electron microscopy were used initially to confirm the presence of crystal proteins in the newly isolated sporulating soil bacteria. Toxicity tests were conducted using crystal proteins from 75 crystal-forming bacterial isolates against A. castellanii trophozoites. The crystal proteins isolated from MA8, CAB7, and AB5 displayed significant antiparasitic activity, with MA8 and CAB7 showing IC50 values of 4.1 and 4.3 µg/mL after 48 hours. SEM analysis revealed morphological differences among the isolates. Phylogenetic analysis indicated the genetic similarity of CAB7 with Bacillus paranthracis, and MA8 with different Bacillus species, potentially indicating that MA8 is a new species. Genome annotation revealed the absence of typical parasporal inclusion genes (Cry or Cyt) but showed the presence of the S-layer protein-encoding gene, which is another group of parasporal inclusions. These findings highlight the presence of little-studied Bacillus paranthracis, and a potentially novel Bacillus species with antiparasitic properties. These genomic insights open avenues for exploring therapeutic applications of CAB7 and MA8 strains.