The wine industry faces growing demand for low- and no-alcohol wines (LNAWs), driven by changing consumer preferences and climate change. The project “INNOWINE”—Cascading Grant Spoke 3 - Project OnFoods—applied an optimised production cycle to limit alcohol formation during fermentation, offering an innovative alternative to conventional post-fermentation dealcoholization. Among the strategies tested, we investigated X-ray irradiation, a non-thermal method proposed to replace sulfur dioxide and improve wine microbial stability. Microbiological quality was assessed in experimental low-alcohol wines (EW) and grape-must permeate (P) versus conventional wine (CW). Total mesophilic prokaryotes and yeasts were quantified using PCA and WLNM, respectively. Putative lactic acid bacteria (LAB), acetic acid bacteria (AAB), and Brettanomyces were enumerated on differential media (i.e. MRS, GYC, mWLNM), all supplemented with cycloheximide to suppress undesired yeasts and improve selectivity. Samples were analysed with and without X-ray treatment at 0.5, 1.0, and 2.0 kGy. Preliminary findings show higher microbial counts in EW and P than CW without irradiation. Irradiation progressively reduced microbial loads from partial reductions (i.e. 0.5 kGy) to microorganism elimination across all media (i.e. 2.0 kGy). X-ray technology appears promising for stabilising LNAW while preserving quality, offering a potential substitute for sulfur dioxide.