We investigated the stability of silver nanoisland films, which were formed on glass surface by the method of out-diffusion, in biocompatible buffers and the applicability of the films in surface enhanced Raman scattering (SERS). We have shown that silver nanoisland films are stable in one of the most widespread in biological studies buffer—phosphate buffer saline (PBS), and in 1:100 water-diluted PBS, in the PBS-based buffer, in which NaCl is replaced by the same amount of NaClO4, and in acidic phosphate buffer. At the same time, the replacement of NaCl in PBS by N(CH3)4Cl leads to the degradation of the nanoislands. It was shown that after exposure to PBS the nanoisland films provided a good SERS signal from a monolayer of 1,2-di(4-pyridyl)ethylene (BPE), which makes silver nanoisland films promising for biosensor applications. Additionally, in our experiments, we registered for the first time that silver nanoparticles formed in the bulk of the samples dissolved after exposing to PBS, while nanoislands on the glass surface stayed unchanged. We associate this phenomenon with the interaction of ions contained in PBS solution with silver, which results in the shift of corresponding chemical equilibrium.