The widespread use of antibiotics causes the development of antibiotic-resistant bacterial strains, which have a severe impact on poultry productivity and human health. As a result, research is continuing to develop safe natural antibiotic alternatives. In the current study, Bacillus pumilus SA388 was isolated from the chicken feces and confirmed to be a probiotic. The selected strain was tested for its antimutagenic and antioxidant capabilities before being employed as a probiotic food supplement and antibiotic alternative. The effect of B. pumilus SA388 impact on broiler chickens' growth performance, gut microbiome, blood biochemical markers, immunological response, and meat quality was also studied. B. pumilus SA388 showed significant bactericidal activity against Streptococcus pyogenes, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella typhi, and Klebsiella pneumonia. A total of 200 chickens were used in the present study, divided equally among four experimental groups (ten birds per group with five replicates): group 1 (control, G1) received a basal diet without B. pumilus SA388, group 2 (G2) received a basal diet supplemented with 0.4 mg/kg of B. pumilus SA388, group 3 (G3) received a basal diet supplemented with 0.8 mg/kg of B. pumilus SA388, and group 4 (G4) received a basal diet supplemented with 1.6 mg/kg of B. pumilus SA388. Over 35 days, the B. pumilus SA388-supplemented groups outperformed the G1 in terms of body weight gain, performance index, and feed conversion ratio, with a preference for the G4 treatment. The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), low-density lipoprotein (LDL), and total cholesterol decreased significantly (P < 0.05) with increasing B. pumilus SA388 dosages compared to the control G1 group. Dietary supplementation of B. pumilus SA388 at 1.6 mg/kg (G4) significantly (P < 0.05) resulted in improved lipid profile, immunological response, thyroid function, and gut microbiota compared to the control group (G1). Compared to the broilers in the control treatment (G1), the addition of B. pumilus SA388 to broilers in G4 significantly (P < 0.05) enhanced juiciness, tenderness, aroma, and taste. Adding B. pumilus SA388 to chicken feed at different doses significantly (P < 0.05) decreased average feed intake while increasing economic and relative efficiency measures. In conclusion, B. pumilus SA388 has been proven to be an effective antibiotic and nutritional supplement.