Biodeterioration represents a major threat to cultural heritage, as microbial colonization can cause both esthetic and structural damage. The use of conventional chemical biocides raises concerns due to environmental and health risks, potential substrate deterioration, and the emergence of resistant strains. In this study, an ozone-loaded bacterial cellulose (OBC) hydrogel was investigated as an eco-friendly, broad-spectrum antimicrobial treatment in the case study of the Cryptoporticus of the Baths of Trajan (Rome, Italy), a hypogean archeological site where some structures are severely affected by phototrophic biofilms. Microorganisms isolated from a colonized wall were employed in laboratory assays. OBC hydrogel exhibited strong antimicrobial activity, with >90% bacterial mortality within 10 min, complete inhibition of fungal spore germination after 24 h, and a marked reduction in microalgal chlorophyll fluorescence comparable to heat-killed controls. Furthermore, tests on Carrara marble and brick specimens artificially contaminated with microalgae confirmed the removal of green staining, restoring surface chromatic parameters (ΔE* < 5) comparable to those obtained with a commercial biocide. In situ applications demonstrated significant suppression of green biofilm for at least two months. These findings support OBC hydrogel as a sustainable, effective, and non-toxic alternative to conventional biocides for controlling microbial and microalgal colonization on cultural heritage surfaces.