Uridine (UdN) is a pyrimidine nucleoside drug that is widely applied in the pharmaceutical industry. The solubility and biological activity of UdN have been enhanced by the formation of inclusion complexes with β-cyclodextrin derivatives (β-CDs), namely, hydroxypropyl-β-cyclodextrin (HPβ-CD) and sulfobutyl-ether-β-cyclodextrin (SBEβ-CD). Optical spectroscopy studies suggested the formation of 1:1 inclusion complexes between UdN and the β-CDs at 303 K, as determined from the Benesi–Hildebrand plot. The solid complexes of UdN and the β-CDs were prepared and characterized by various spectroscopic techniques, and the results indicated the inclusion of UdN in the β-CD cavity. The energetically favored binding interaction of UdN with the β-CDs was confirmed by molecular modeling studies. In addition, the investigations on the antibiofilm and antioxidant activities indicated that the β-CD/UdN complexes exhibited better fungicidal, cytotoxic, and cellular uptake activities than UdN.