Unprecedented gold(i) bioconjugates bearing non-proteinogenic amino acid 4-mercaptoproline species as bioorganic ligands have been prepared. Firstly, the synthesis of Boc-Pro(SH)-OMe (1) has been accomplished by standard procedures. The subsequent reaction of 1 with [AuCl(PR3)] gives complexes Boc-Pro(SAuPR3)-OMe (PR3 = PPh3 (2), PPh2Py (3)). Starting from complex 2 several structural modifications have been performed, in addition to the incorporation of a different phosphine in 3, such as the formation of the acid Boc-Pro(SAuPPh3)-OH (4), the synthesis of a dipeptide derivative by coupling the amino acid glycine tert-butyl ester Boc-Pro(SAuPPh3)-Gly-O(t)Bu (5), or the coordination of another gold phosphine fragment to the sulfur atom as in [Boc-Pro(SAuPPh3)2-OMe]OTf (6). The cytotoxic activity in vitro of these complexes has been evaluated against three different tumor human cell lines, A549 (lung carcinoma), Jurkat (T-cell leukaemia) and MiaPaca2 (pancreatic carcinoma). All the complexes displayed excellent cytotoxic activity with IC50 values in the low μM range and even in the nM range in some cases. Structure-Activity Relationships (SAR) observed from this family of complexes opens the possibility of designing more potent and selective promising gold(i) anticancer agents.