Siderophoregenic Bacillus strain DET9 has been selectively isolated from dairy waste. It was evaluated for probiotic characteristics and susceptibility pattern against antibiotics. Its spores showed excellent tolerance to simulated gastrointestinal tract conditions and exhibited antimicrobial activity against organisms such as Escherichia coli, Micrococcus flavus, and Staphylococcus aureus. Its susceptibility to antibiotics reduces the prospect to donate resistance determinants if administered in the form of probiotic preparations. It was observed to produce similar to 60 mg/l catecholate type of siderophore under iron stressed conditions, identified as a 2,3-dihydroxy benzoic acid by high-performance liquid chromatography, infrared spectroscopy, nuclear magnetic resonance, and mass spectral analysis. Partial 16S-rRNA gene sequencing analysis shows that the isolate exhibited homology with Bacillus thuringiensis and Bacillus weihenstephanensis, whereas biochemical characterization revealed its novelty. DET9 exhibited no mortality of fishes in a 60-day trial, when fishes (surfi tetra) were challenged up to 100 ppm cell concentration, with their daily diet.