Strawberries belonging to cultivar Clery (Fragaria x ananassa (Duchesne ex Weston)), cultivated in central Italy were subjected to a multi-methodological experimental study. Fresh and defrosted strawberries were exposed to different processing methods, such as homogenization, thermal and microwave treatments. The homogenate samples were submitted to CIEL*a*b* color analysis and Head-Space GC/MS analysis to determine the impact of these procedures on phytochemical composition. Furthermore, the corresponding strawberry hydroalcoholic extracts were further analyzed by HPLC-DAD for secondary metabolites quantification and by means of spectrophotometric in vitro assays to evaluate their total phenolic and total flavonoid contents and antioxidant activity. These chemical investigations confirmed the richness in bioactive metabolites supporting the extraordinary healthy potential of this fruit as a food ingredient, as well as functional food, highlighting the strong influence of the processing steps which could negatively impact on the polyphenol composition. Despite a more brilliant red color and aroma preservation, non-pasteurized samples were characterized by a lower content of polyphenols and antioxidant activity with respect to pasteurized samples, as also suggested by the PCA analysis of the collected data.