Microbial management is one of the most critical aspects of winery operations and is normally achieved via chemical control. This study sought to optimize winery cleaning and sanitation protocols for the management of winery spoilage microorganisms by applying multiple techniques commonly found in clinical health settings to winery-relevant conditions. The minimum inhibitory concentration and minimum biocidal concentration assay and a modified minimum biofilm inactivation assay were performed for three common winery spoilage yeast (S. cerevisiae, B. bruxellensis, Z. baili). Results indicate that inhibitory and biocidal concentrations vary dramatically between organisms but are largely in line with established application rates for inactivation of all cells in planktonic and biofilm physiologies. Dual-channel fluorescence staining was employed to determine minimum inactivation time for S. cerevisiae using two peracetic acid concentrations. Propidium Iodide and SYBR Green 1 stains were validated as a live/dead proxy (R2 = 0.99) and used to determine the contact time required to inactivate cell suspensions. Peracetic acid treatment trials indicate that S. cerevisiaepopulations are inactivated in five minutes or less at concentrations of 1−1.5 mg/L. In conjunction, these experiments provide insight for winemakers to critically think about cleaning and sanitation protocols and how to optimize these processes.