Abstract Background Healthcare-associated infections (HAIs) caused by microbes that acquire antimicrobial resistance (AMR) represent an increasing threat to human health worldwide. The high use of chemical disinfectants aimed at reducing the presence of pathogens in the hospital environment can simultaneously favor the selection of resistant strains, potentially worsening AMR concerns. In the search for sustainable ways to control bioburden without affecting this aspect, probiotic-based sanitation (PBS) using Bacillus spp. was proposed to achieve stable reduction of pathogens, AMR, and associated HAIs. Although Bacillus probiotics are classified as nonpathogenic, comprehensive data about the potential genetic alterations of these probiotics following prolonged contact with surrounding pathogens are not yet available. This study aimed to assess in depth the genetic content of PBS-Bacillus isolates to evaluate any eventual variations that occurred during their usage. Results WGS analysis was used for the precise identification of PBS-Bacillus species and detailed profiling of their SNPs, resistome, virulome, and mobilome. Analyses were conducted on both the original PBS detergent and 172 environmental isolates from eight hospitals sanitized with PBS over a 30-month period. The two species B. subtilis and B. velezensis were identified in both the original product and the hospital environment, and SNP analysis revealed the presence of two clusters in each species. No virulence/resistance genes or mobile conjugative plasmids were detected in either the original PBS-Bacillus strain or any of the analyzed environmental isolates, confirming their high genetic stability and their low/no tendency to be involved in horizontal gene transfer events. Conclusions The data obtained by metagenomic analysis revealed the absence of genetic sequences associated with PBS-Bacillus and the lack of alterations in all the environmental isolates analyzed, despite their continuous contact with surrounding pathogens. These results support the safety of the Bacillus species analyzed. Further metagenomic studies aimed at profiling the whole genomes of these and other species of Bacillus, possibly during longer periods and under stress conditions, would be of interest since they may provide further confirmation of their stability and safety.