| Summary: | Severe ocular infections by <i>Acanthamoeba</i> sp. lead to keratitis, resulting in irreversible vision loss in immune-compromised individuals. When a protozoal infection spreads to neural tissues, it causes granulomatous encephalitis, which can be fatal. Treatment often takes longer due to the transition of amoeba from trophozoites to cyst stages, cyst being the dormant form of <i>Acanthamoeba</i>. A prolonged use of therapeutic agents, such as ciprofloxacin (Cipro), results in severe side effects; thus, it is critical to improve the therapeutic efficacy of these widely used antibiotics, possibly by limiting the drug-sensitive protozoal-phase transition to cyst formation. Owing to the biomedical potential of selenium nanoparticles (SeNPs), we evaluated the synergistic effects of ciprofloxacin and <i>Rhizobium pusense</i>–biogenic SeNPs combination. SeNPs synthesized using <i>Rhizobium pusense</i> isolated from root nodules were characterized using UV–Visible spectrophotometer, FT-IR, SEM with EDX, particle size analysis, and Zeta potential. The combination was observed to reduce the sub-lethal dose of Cipro, which may help reduce its side effects. The selenium and ciprofloxacin (SeNPs–Cipro) combination reduced the LC<sub>50</sub> by 33.43%. The anti-protozoal efficacy of SeNPs–Cipro was found to transduce through decreased protozoal-cyst formations and the inhibition of the galactosidase and protease enzymes of trophozoites. Furthermore, high leakage of sugar, proteins, and amino acids during the SeNPs–Cipro treatment was one primary reason for killing the trophozoites. These experimental results may be helpful in the further pre-clinical evaluation of SeNPs–Cipro to combat protozoal infections. Future studies for combinations of SeNPs with other antibiotics need to be conducted to know the potential of SeNPs against antibiotic resistance in <i>Acanthamoeba.</i>
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