The 1999–2000 UC Sampdoria season saw the club finish 5th in Serie B, a point away from promotion.

Sampdoria's kit was manufactured by Japanese company Asics and sponsored by Japanese gaming company Sega, who used their sponsorship to promote their console Dreamcast.

Squad at end of season

Note: Flags indicate national team as defined under FIFA eligibility rules; some limited exceptions apply. Players may hold more than one non-FIFA nationality.

No. Pos. Nation Player — GK  ITA Christian Puggioni — GK  ITA Matteo Sereni — DF  ITA Marcello Castellini — DF  ITA Emanuele Pesaresi — DF  ITA Vittorio Tosto — DF  POR Hugo — DF  YUG Nenad Sakić — DF  URU Damián Macaluso — MF  ITA Stefano Casale — MF  ITA Davide Dionigi — MF  ITA Fabrizio Ficini — MF  ITA Vincenzo Iacopino

No. Pos. Nation Player — MF  ITA Emanuele Matzuzzi — MF  ITA Marco Sanna — MF  ITA Marco Sgrò — MF  ITA Guglielmo Stendardo — MF  ITA Gaetano Vasari — MF  ITA Simone Vergassola — MF  YUG Bratislav Živković — MF  BRA Doriva — FW  ITA Carmine Esposito — FW  ITA Francesco Flachi — FW  ITA Francesco Palmieri — FW  YUG Zoran Jovičić

Note: Flags indicate national team as defined under FIFA eligibility rules; some limited exceptions apply. Players may hold more than one non-FIFA nationality.

No. Pos. Nation Player — GK  ITA Federico Bigliazzi (to Lucchese)

No. Pos. Nation Player — DF  ITA Alessandro Grandoni (on loan to Torino)