The colonization of stone-built monuments by different organisms (algae, fungi, lichens, bacteria, and cyanobacteria) can lead to biodeterioration of the stone, negatively affecting the artistic value of the heritage. To address this issue, laser cleaning has been widely investigated in recent years, due to the advantages it offers over traditional mechanical and chemical methods: it is gradual, selective, contactless, and environmentally friendly. That said, the laser parameters should be optimized in order to avoid any by-effects on the surface as a result of overcleaning. However, as the adjustment of each parameter to clean polymineralic stones is a difficult task, it would be useful to know the effect of overcleaning on the different forming minerals depending on the wavelength used. In this paper, three different wavelengths (355 nm, 532 nm, and 1064 nm) of a Q-Switch neodymium-doped yttrium aluminum garnet (Nd:Y3Al5O12) laser, commonly known as QS Nd:YAG laser were applied to extract a naturally developed sub-aerial biofilm from Vilachán granite, commonly used in monuments in the Northwest (NW)Iberian Peninsula. In addition to the removal rate of the biofilm, the by-effects induced for fluences higher than the damage threshold of the stone were evaluated using stereomicroscopy, color spectrophotometry, and scanning electron microscopy with energy-dispersive x-ray spectroscopy. The results showed that different removal rates were obtained depending on the wavelength used and 532 nm obtained the highest removal level. In terms of by-effects, biotite melting was registered on all surfaces regardless of the wavelength. In addition, 532 nm seemed to be the most aggressive laser system, inducing the greatest change in appearance as a result of extracting the kaolinite crackled coating and the segregations rich in Fe, which are a result of natural weathering. These changes were translated into colorimetric changes visible to the human eye. The surfaces treated with 355 nm and 1064 nm showed lower surface changes.