A 17th century wall painting representing a Virgin between two Saints in a noble Italian renaissance palace, Palazzo Gallo in Bagnaia (Viterbo, Italy), was restored in 2021 in the context of a wider restoration campaign interesting the main room of the palace built by cardinal Sansoni Riario. Diagnostic analyses done with traditional characterization techniques (optical microscopy on micro-stratigraphic sections, X-ray fluorescence spectroscopy and Fourier transform infrared spectroscopy) provided the identification of both original painting and restoration materials, while imaging investigations as ultraviolet fluorescence photography, false color images and multispectral mapping provided by hypercolorimetric multispectral imaging (HMI) technique enabled the evaluation of the state of conservation, locating restoration interventions and supporting the monitoring of the cleaning procedure. An altered protective Paraloid-based coating dating from early 2000s had to be removed due to the unpleasant glossy finishing given to the painted surface, making the scene barely readable. To pursue a restoration protocol based on environmental sustainability and green chemistry, enzyme-based gels marketed by Nasier-Brenta© and CTS© companies were tested in different protocols for the cleaning of the mash covering the painting. Although some interesting results were observed, the enzymatic cleaning had a scarce effectiveness with timing beyond a reasonable interval. Traditional chemical solvents as Dowanol PM (methoxy-propanol) and benzyl alcohol were necessary to complete the cleaning of the painting surface.