Bacillus species isolated from honeybee Apis mellifera gut, honey and bee bread samples were characterized for their in vitro probiotic and safety attributes. Alpha and γ haemolytic cultures were tested for their antibiotic resistance, antibacterial spectrum, acid and bile tolerance, adhesion ability (auto‐aggregation, co‐aggregation and hydrophobicity) and phenol tolerance. Safety criteria included evaluation of virulence genes and cytotoxicity percentages. Bacillus isolates inhibited both Gram‐positive and Gram‐negative pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and Streptococcus mutans, while none could inhibit Listeria monocytogenes. Among the isolates, Bacillus subtilis ZH05, ZB03 and ZG025 showed resistance to most of the tested antibiotics and were considered unsafe. B. subtilis (4) and B. licheniformis (1) tolerated acidic pH and bile conditions, never the less were more tolerant in simulated intestinal conditions vis‐a‐vis gastric conditions. In 0·5% phenol concentrations, B. licheniformis ZH02 showed highest growth, while, B. subtilis ZG029 demonstrated highest auto‐aggregation (65 ± 4·6) and hydrophobicity (23 ± 3·6) percentages (P < 0·05). The isolates lacked virulence genes (hblA, hblC, hblD, nhe, cytK and ces), and their cytotoxic percentage on Caco‐2 cell lines was ˂15%. Overall, honeybees appear to be a good source of Bacillus species exhibiting typical in vitro probiotic properties, which could be of commercial interest.

European foulbrood (EFB), a disease affecting larvae of honey bees, Apis mellifera L. (Hymenoptera: Apidae), is caused by the Gram-positive bacterium Melissococcus plutonius corrig. (ex White) Bailey & Collins (Vasquez et al., 2012). Other secondary invaders like Paenibacillus alvei (Cheshire & Cheyne) Ash et al., Brevibacillus laterosporus (Laubach) Shida, and Enterococcus faecalis (Andrewes & Horder) Schleifer & Kilpper-Bälz are also associated with EFB (Pietropaoli et al., 2022). This bacterial disease is responsible for honey bee colony decline and consequently