Background: Bacillusspecies are widely distributed in all environments including health care settings and represent one of  the highly resistant organisms. Objective: This study aimed to find out the prevalence, molecularcharacterization of genetic diversity among studied Bacillus species in Egyptian hospitals environment and their antibiotic susceptibility profile. Methods: A total 528 swab samples were collected from different hospitals environment. Isolation and identification were performed according to conventional bacteriological methods, semi-automated and molecular characterization methods. Antimicrobial susceptibility was carried against different groups of antimicrobial agents. Results: The most isolated microorganism was Bacillus spp. (43.2%), followed by coagulase-negative Staphylococci (19.2%), Staphylococcus aureus (15.2%), Enterococus spp. (10.1%), Gram-negative rods (8.9%), and Micrococcus spp. (3.4 %). The most prevalent species, were Bacillus cereus (46.6%) followed by Bacillus subtilus (38.1%) while, Bacillus pumilus was the least (1.1%). A majority of Bacillus isolates (25.6%) were isolated in Internal medicine department followed by Emergency department (18.8%) while operating rooms showed the lowest prevalence rate (4.5%). Antimicrobial susceptibility testing revealed high resistance of Bacillus isolates to β-lactams and tetracycline antibiotics. Multi-drug resistance (MDR) isolates which resistance to three or more antibiotics was (21.6%). Susceptibility reports of the 176 Bacillus isolates revealed 45 antibitypes and the most common was antibiotype 31, which included 32 isolates (18.2%), that is resistant to both penicillin and cefoxitin. Conclusions: This study revealed that, dissemination of Bacillus species in study hospital environments with high resistance to β-lactams and tetracycline antibiotics. The molecular analysis revealed the existence of genetic diversity among studied Bacillus isolates. Thus, monitoring the hospital environment is an important tool in the prevention of hospital-associated infection by Bacillus species.