The ophiolitic rocks of the Attic-Cycladic crystalline belt are considered of key importance for understanding the Mesozoic tectonic evolution of the Aegean region. Unresolved questions concern their tectono-stratigraphic relationships across the region. The mode of occurrence of the Cycladic ophiolites varies, as they appear as: (a) dismembered blocks (olistoliths) within the supra-detachment units of Paros and Naxos; (b) mélange formations in the upper structural unit of western Samos and Skyros; and (c) meta-ophiolitic mélange in the Cycladic Blueschist Unit (CBU) from central Samos. The trace element geochemistry and Pb-Sr-Nd isotopes of the mafic ophiolitic rocks indicate four geochemical groups: (a) the upper unit metabasites from Paros and western Samos (Kallithea) display an evolved basaltic composition (Mg# 40.2–59.6), with low Zr/Nb values (5–16) and high Ce/Y values (1.3 to 2.6) compared to MORB, indicating island-arc tholeiite affinities; (b) Naxos upper unit metabasalts show spider diagrams patterns indicating ocean island basalt (OIB-type) affinities; (c) Central Samos metagabbros (CBU) are primitive rocks with Back-Arc Basin basalt affinities; (d) the Skyros metadolerites and Tinos (Mt Tsiknias) and S. Evia (CBU) metagabbros, cluster as a separate geochemical group; they exhibit high MgO values (>10 wt %), very low TiO2 values (0.1–0.2 wt %), Y and Yb, and depleted trace element N-MORB normalized patterns, similar to volcanic rocks formed in modern oceanic fore-arc settings, such as boninites. A combination of the Pb- and Sr-isotopic compositions of Cycladic metabasites indicate that the Pb and Sr incorporated in the Cycladic ophiolites correspond to mixtures of magmatic fluids with seawater (206Pb/204Pb = 18.51–18.80; 207Pb/204Pb = 15.59–15.7; 208Pb/204Pb = 39.03–39.80 and initial 87Sr/86Sr80 = 0.705–0.707). Furthermore, peridotite relicts from Samos, Paros, and Naxos—irrespective of the structural unit—display chemical affinities of ocean floor peridotites formed in a supra-subduction zone. The characteristics of harzburgite relicts in Cycladic serpentinites and Skyros indicate a highly residual nature of the mantle source. Geochemical data from this study shed further light on the complex structure of the oceanic lithosphere from which the Cycladic ophiolites originated.