Alicyclobacillus sp. are acidothermophilic bacteria frequently contaminating fruit based products (juices and juice concentrates). These sporulating bacteria are able to survive at elevated temperatures and highly acidic environments which causes difficulties in their removal from industrial environments. Although numerous literature data examine Alicyclobacillus sp. presence in fruit based products and methods of their elimination, there is still a limited knowledge on ability of these bacteria to adhere to abiotic surfaces. Therefore, the objective of this study was to determine Alicyclobacillus sp. cells' hydrophobicity and capability of biofilm formation on a glass surface. The degree of cells hydrophobicity, according to Microbial Adhesion to Hydrocarbon (MATH) and Salt Aggregation Test (SAT), was investigated for eleven environmental isolates from natural Polish habitats, identified as Alicyclobacillus sp., and a Alicyclobacillus acidoterrestris DSM 3922 reference strain. The dynamics of biofilm formation within 3-day incubation on a glass surface was evaluated and quantified by a plate count method both, for cultures with and without agitation. All of the bacterial strains tested expressed ability to colonize a glass surface and four environmental isolates were classified as fast-adherent strains. The mature biofilm structures were predominantly formed after 48 hours of incubation. Dynamic culturing conditions were observed to accelerate the biofilm formation. The majority of strains expressed a moderate hydrophobicity level both, in SAT (41.7%) and MATH-PBS (75.0%), as well as MATH-PUM (91.7%) tests. However, no correlation between hydrophobicity and cell adherence to a glass slide surface was observed.