Probiotics are living microorganisms when administered in adequate amounts confer health benefits to the host. In the present study, a soil isolate was identified as Bacillus subtilis based on the 16S rRNA sequencing. In probiotic functional characterization (in vitro), B. subtilis SKB/2074 produced 10 different enzymes, was stable under simulated gastric conditions (pH 2.5/1-3 hr), bile salt (0.05-0.3% w/v), and temperature (40-90 degrees C) conditions. B. subtilis SKB/2074 cells were non-hemolytic, found susceptible to the 30 antibiotics, and showed antimicrobial activity against Escherichia coli, Salmonella typhimurium, and Clostridium perfringens. In in vivo studies, B. subtilis SKB/2074 demonstrated encouraging results to reverse E. coli and castor oil incited diarrhea in Wistar rats and Albino mice, respectively. Histopathological studies exhibited restoration of damaged mucosal epithelium cells and recovers veracity of goblet cells (colon). B. subtilis SKB/2074 exhibited immunomodulatory effects (increased immunoglobulins in blood and weight of spleen and thymus) and significant antioxidant activity (84.14%), reducing capacity and ascorbate auto-oxidation inhibition effect (95.13%). In poultry field studies, B. subtilis SKB/2074 significantly improved growth performance and lowered mortality rate in broiler chickens. Based on these preliminary scientific assessments B. subtilis SKB/2074 is likely to be used as potential probiotic and antidiarrheal agent in humans and animal healthcare.