The Oriented architecture of macromolecules plays a critical role in many aspects of Nanobiotechnology such as in the development of biosensors. To this regard, S-layers which constitute the outermost cell envelope component of many prokaryotic organisms, represent unique self assembled systems with the capability to rearrange into monomolecular and oriented arrays. These properties can be exploited to promote their crystallization on surfaces (e.g. silicone) which is pivotal for the subsequent immobilization of macromolecules and development of new biosensors. In this work the crystallization of bacterial S-layers obtained from Bacillus thuringiensis and Bacillus sphaericus CCM2177 on silicone, mica and quartz crystal surfaces were investigated. The SDS page results of S-layers isolated from the above mentioned bacteria put in evidence that their molecular weight (MW) was around 120 KDa and, as reported in the literature, slightly higher for those extracted by Bacillus thuringiensis. In addition, results showed that S-layers isolated from Bacillus thuringiensis form large crystalline domains on mica after 5 min whereas those extracted from Bacillus sphaericus CCM 2177 form a compact monolayer on silicone after 2 h. Results in this work put in evidence the possibility to use these substrates for the fabrication of sensitive biosensors.