This study evaluated changes in physicochemical characteristics, bioactive compounds, in vitro antioxidant activity, and antimicrobial activity during four ripening stages (M0%, M50%, M80%, and M100%) of ‘Castilla’ blackberry, ‘Buckingham Tayberry’ raspberry, ‘Heritage’ raspberry, blueberry, and ‘Mortiño’. Bioactive compounds were quantified using rapid-resolution liquid chromatography, antioxidant activity was determined using the ABTS and DPPH assays, and antimicrobial activity was evaluated through minimum inhibitory concentration assays against 13 bacteria and 7 fungal strains. Ripening effects were fruit- and compound-dependent. Soluble solids generally increased, and titratable acidity decreased during ripening. Early stages were characterised by higher concentrations of several organic and phenolic acids and, in some fruits, greater radical scavenging and antimicrobial responses. Advanced ripening favoured the accumulation of carotenoids and anthocyanins, particularly in blueberry and ‘Mortiño’. Antimicrobial activity also varies according to fruit type, ripening stage, and microorganism. ‘Mortiño’ showed the lowest MIC against Enterococcus faecalis, whereas the other fruits displayed selective inhibition against different bacterial and fungal strains. Overall, no single ripening stage maximised all compositional and biological variables, demonstrating that harvest stage selection should be fruit- and purpose-specific.