Comfrey (Symphytum officinale L.) roots are well-known bioactive ingredients included in various cosmeceutical and pharmaceutical preparations. In this study, the influence of the post-harvest storage on the chemico-biological potential of roots collected from different European regions and stored for up to six months was investigated. Total phenolic content (TPC) and total phenolic acid content (TPAC) were spectrophotometrically estimated, whereas the levels of individual phenolic and pyrrolizidine alkaloidal markers were determined by HPLC-DAD and HPLC-MS/MS, respectively. The changes in the biological potential was tracked via antioxidant (DPPH, ABTS, CUPRAC, and FRAP) and anti-enzymatic (cholinesterase, tyrosinase, glucosidase, and amylase) assays. TPC and TPAC varied from 6.48–16.57 mg GAE/g d.w. root and from 2.67–9.03 mg CAE/g, respectively. The concentration of the four phenolics (rosmarinic acid, globoidnan A, globoidnan B, rabdosiin) and six pyrrolizidine alkaloids generally showed maximum values at 1–3 months, after which their levels significantly decreased. With respect to the bioassays, the samples showed a wide range of antioxidant and anti-enzymatic effects; however, a direct storage time–bioactivity relationship was not observed. Similar conclusions were also revealed by the multivariate and correlation analyses. Our study could improve the current knowledge of the shelf-life properties of comfrey-based products and enhance their industrial exploitation.