Abstract Aims This study reports the results of the application of a new agar-gauze biogel system activated with viable bacterial cells to altered wall paintings. Methods and Results Biocleaning using agar biogel and agar-gauze biogel systems was performed onsite by direct application to altered wall painting surfaces (25–1000 cm2). The treatments were performed for the restoration of two original Italian sites: (i) at the Vatican Museums, Cristo che salva Pietro dalle acque—La Navicella, a wall painting by Giovanni Lanfranco (1627–1628) and (ii) at Pisa Cathedral Cupola, Incarnato, a wall painting by Orazio Riminaldi (1593–1630) and his brother Girolamo Riminaldi. The novelty of this study is the use of viable Pseudomonas stutzeri A29 cells in an advanced agar-gauze biogel system and the short bio-application contact times of between 3 and 12 h. The historical artworks were altered by lipid and protein residues from past restoration, as confirmed by Py-gas chromatography–mass spectrometry and FT-IR data. The effectiveness of the biological treatment was assessed, and general considerations were discussed. Conclusions The short bio-application contact time of advanced agar-gauze gel activated with viable P. stutzeri cells makes this biotechnology promising as an alternative method to the traditional onsite cleaning techniques currently in use for altered historical wall paintings. Significance and Impact of the Study In this study, we report for the first time the biocleaning of altered materials located in vertical and vaulted areas using agar-gauze biogel with short application times. These findings are of great significance for future restoration activities and are crucial for determining the best preservation strategies in this field.