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This study aimed to assess the counts and biodiversity characterization of aerobic sporeforming bacteria (ASB) in powdered infant formula (PIF). Fifty-four (n = 54) samples of PIF of three brands were analyzed for mesophilic aerobic bacteria, and ASB counts. ASB isolated from PIF were assessed for their ability to produce spoilage enzymes and hemolytic activity and further identified by MALDI-TOF mass spectrometry. Then, the isolates were subjected to rpoB gene typing and assessment of bceT, entFM, nhe (A, B, C), and hbl (A, B, C) toxin genes. The main species isolated were B. licheniformis (54%), followed by B. cereus (33%) and B. subtilis (5%). The ASB counts ranged from 1 to 4 log CFU/g, and the mean was 2.9 log CFU/g for mesophilic aerobic sporeforming bacteria (MSC) and 2.5 log CFU/g for thermophilic aerobic sporeforming bacteria (TSC). Most PIF samples presented MSC and TSC counts between 2 and 3 log CFU/g. A total of 13%, 50%, and 37% of the samples presented MSC counts from <2 log CFU/g, between 2 and 3 log CFU/g and between 3 and 4 log CFU/g, respectively. Among the ASB isolates, 97% had protease, 84% hydrolyzed starch, 66% had hemolytic activity, and 61% had lecithinase activity. A total of 44 out of 120 isolates harbored at least one toxin gene; 56% for B. cereus, 34% for B licheniformis, and less than 5% for B. subtilis, B pumilus, and L. sphaericus. All B. cereus isolates harbored the nhe gene, 60% entFM, 44% cytK, 32% bceT, and 28% hbl genes. Besides, 17% of B. licheniformis harbored nhe. A small proportion of B. subtilis, B. pumilus, and L. sphaericus carried toxin genes. The rpoB based phylogenetic tree provided high resolution among Bacillus species. The findings of this study provide insights into the phenotypic and genotypic biodiversity of Bacillus present in PIF., (Copyright © 2022 Elsevier B.V. All rights reserved.)