Electrospray ionization mass spectrometry (ESI-MS) was successfully employed for the identification of six nitrido technetium mixed ligand complexes with a general formula of [^99gTc(N)(O,S-BID)(PNP)], where PNP represents a heterodiphosphine and O,S-BID represents a simple dianionic bidentate ligand (compounds 1–3) or a more sophisticated N-substituted O,S-cysteine framework conjugated with a bio-active molecule (BAM) (compounds 4–6). In spite of similar coordination spheres exhibited by all the complexes investigated, simple co-ordination compounds 1–3 displayed collisionally-induced fragmentation processes (MSn) different from those observed in biomolecule-containing compounds 4–6. In the latter, more decomposition channels were observed. This behavior is likely to be associated with some additional intramolecular contacts of the biomolecule (or part of the biomolecule) with pendant group(s) incorporated in the PNP-co-ligand. This view is further supported by the observations arising from both in vitro binding affinity experiments and nuclear magnetic resonance investigations. The presence of cationized forms for all compounds 1–6 and the practical lack of the [2M + Na]⁺ species for biomolecule-containing compounds 4–6 provided further evidence of a subtly different structural conformation.