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Characterizing the anchoring properties of smectic liquid crystals (LCs) in contact with bacterial solutions is crucial for developing biosensing platforms. In this study, we investigate the anchoring properties of a smectic LC when exposed to Bacillus subtilis and Escherichia coli bacterial suspensions using interfaces with known anchoring properties. By monitoring the optical response of the smectic film, we successfully distinguish different types of bacteria, leveraging the distinct changes in the LC’s response. Through a comprehensive analysis of the interactions between bacterial proteins and the smectic interface, we elucidate the potential underlying mechanisms responsible for these optical changes. Additionally, we introduce the utilization of topological defects, the focal conic domains (FCDs), at the smectic interface as an indicative measure of the bacterial concentration. Our findings contribute to the understanding of bacteria–LC interactions and demonstrate the significant potential of smectic LCs and their defects for biosensing applications, paving the way for advancements in pathogen detection and protein-based sensing.