The antibacterial properties of ultrafine, highly crystalline ¹⁰B-enriched hexagonal boron nitride nanoparticles (¹⁰BNNPs: synthesized at 1400 °C within a growth duration of 150 min) against the gram-positive bacterium (Actinomycete, and Streptococcus pyogenes), and the gram-negative bacterium (Klebsiella pneumonia, and Enterobacter) are the main focus of the current investigation. ¹⁰BNNPs are found more responsive against Actinomycete and Klebsiella pneumonia, and exhibit the largest zone of inhibition at all tested concentrations. On a concentration of 100 μg/ml particles, it has been observed that ¹⁰BNNPs provide a maximum zone of inhibition of 30 mm, and 25 mm for Actinomycete, and Klebsiella pneumonia, and a minimum zone of inhibition of 16 mm, and 14 mm for Streptococcus pyogenes, and Enterobacter, respectively. The results suggest that the synthesized ¹⁰BNNPs can be a potential alternative to the existing antibiotics against Actinomycete and Klebsiella pneumonia bacteria in the future.