Thiolate-protected gold nanoclusters (AuNCs) of sub-2 nm size have been synthesized through a novel bottom-up approach using the organometallic precursor [Au(CF)(tht)] (tht = tetrahydrothiophene) in a one-pot reaction under mild conditions. This protocol is simple, rapid (1 h), versatile (applicable to thiolate ligands of varying molecular sizes), and reproducible, yielding AuNCs with low size dispersion. Furthermore, the resulting nanomaterials exhibited remarkable catalytic activity, effectively reducing the pollutant 4-nitrophenol to 4-aminophenol, as well as promising photothermal and photodynamic properties upon exposure to an 808 nm laser, converting light into thermal energy and generating reactive oxygen species (ROS). Additionally, AuNCs stabilized with a nonapeptide demonstrated efficient catalase-like activity, thereby potentially enhancing the efficacy of photodynamic therapy. The cytotoxic effects against cancer (HeLa) and healthy cells (HDF) were also evaluated, showing greater selectivity for HeLa cells, with higher toxicity and increased ROS generation.