Stone surfaces are colonized by subaerial biofilms (SABs), complex multicellular communities embedded in a self-produced matrix of hydrated extracellular polymeric substances (EPS). Recent studies have shown that the SABs on artistic surfaces are not only detrimental, leading to biodeterioration, but they can also have a protective role. This is especially true for porous stones, where the SABs form a protective interface layer between the lithic material and the surrounding environment. This study reproduced mono and dual-species SABs on limestone coupons to examine stone surface properties with both living and dead SABs under moist (75% RH) and dry (30% RH) conditions. The objectives were twofold: (1) to study how live SABs impact surface water sorption and wettability in short-term experiments under different environmental conditions and (2) to explore whether dead SABs influence surface water dynamics as much as live SABs in long-term experiments. Well-known, non-invasive analyses were employed to characterize the samples, such as microscopic observations, contact angle measurements, colorimetric and water content evaluation, and a specific Infrared thermography analysis called Spilling Drop Test. The analyses were carried out before and after exposing the biofilms to different environmental conditions, including changes in relative humidity (RH) or exposure time; the results showed that SABs modified the water sorption of materials, creating a layer at the stone-air interface. These findings open new opportunities to harness microbiological growth for protecting stone surfaces from environmental stressors. Unlike traditional conservation strategies that rely on synthetic hydrophobic coatings—which are often associated with material incompatibility, long-term degradation, and environmental concerns—SABs may act as an eco-friendly, natural barrier against moisture penetration and weathering in limestone.