Proteins and organothiols are known for their high binding affinity to noble metal surface including gold nanoparticles (AuNPs). Numerous reports have been dedicated to AuNP interaction with protein or organothiol alone. Competitive protein and organothiol (OT) interaction is, however, mostly an unexplored area. The research reported here focused on developing a fundamental understanding of sequential and simultaneous protein and organothiol interaction with AuNPs in which protein and OT are added either simultaneously or sequentially into the colloidal AuNP solutions. In studies of OT interactions with bovine serum albumin (BSA) stabilized AuNPs, we found that the protein coating layer is highly porous and permeable for small molecules such as mercaptobenzimidazole (MBI), cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). Based on the amounts of MBI adsorbed and the kinetics of MBI adsorption onto BSA stabilized AuNPs, we were able to get an insight into protein conformational changes on the AuNPs. The competitive and sequential studies of protein and OT interactions with AuNPs involving eight model organothiols showed that the protein and OT cosorption onto AuNPs is a kinetically controlled process. The AuNP stability against ligandsorption-induced AuNP aggregation differed significantly among the AuNP/OT and AuNP/BSA/OT mixtures where the AuNP stability order increased from (AuNP/OT)/BSA to AuNP/(BSA/OT), and finally (AuNP/BSA)/OT samples (the two components inside the parenthesis are mixed first followed by the addition of the third component). The studies on the role of cysteine in protein-AuNP interactions found that the cysteine has no significant effect on the kinetics of protein adsorption onto AuNPs. However the stability of the protein-AuNP complex against the organothiolsorption induced AuNP aggregation increased as the number of cysteine residues increased from zero to two. Besides providing new insights on protein interaction with AuNPs, this research is important for AuNP biological/biomedical applications because AuNPs in biofuids encounter a mixture of proteins and OTs in addition to other molecular species.