Prostate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)-targeted radiotracers, for example, Tc-labeled PSMA tracer analogs for radioguided surgery. The purpose of this study was to develop a second-generation Tc-labeled PSMA-targeted tracer incorporating a fluorescent dye. Several PSMA-targeted hybrid tracers were synthesized: glutamic acid-urea-lysine (EuK)-Cy5-mas , EuK-(SO )Cy5-mas , EuK-Cy5(SO )-mas , EuK-(Ar)Cy5-mas , and EuK-Cy5(Ar)-mas ; the Cy5 dye acts as a functional backbone between the EuK targeting vector and the 2-mercaptoacetyl-seryl-seryl-seryl (mas ) chelate to study the dye's interaction with PSMA's amphipathic entrance funnel. The compounds were evaluated for their photophysical and chemical properties and PSMA affinity. After radiolabeling with Tc, we performed in vivo SPECT imaging, biodistribution, and fluorescence imaging on BALB/c nude mice with orthotopically transplanted PC346C tumors. The dye composition influenced the photophysical properties (brightness range 0.3-1.5 × 10 M × cm ), plasma protein interactions (range 85.0% ± 2.3%-90.7% ± 1.3% bound to serum, range 76% ± 0%-89% ± 6% stability in serum), PSMA affinity (half-maximal inhibitory concentration IC range 19.2 ± 5.8-175.3 ± 61.1 nM) and in vivo characteristics (tumor-to-prostate and tumor-to-muscle ratios range 0.02 ± 0.00-154.73 ± 28.48 and 0.46 ± 0.28-5,157.50 ± 949.17, respectively; renal, splenic, and salivary retention). Even though all tracer analogs allowed tumor identification with SPECT and fluorescence imaging, Tc-EuK-(SO )Cy5-mas had the most promising properties (e.g., half-maximal inhibitory concentration, 19.2 ± 5.8, tumor-to-muscle ratio, 5,157.50 ± 949.17). Our findings demonstrate the intrinsic integration of a fluorophore in the pharmacophore in PSMA-targeted small-molecule tracers. In this design, having 1 sulfonate on the indole moiety adjacent to EuK ( Tc-EuK-(SO )Cy5-mas ) yielded the most promising tracer candidate for imaging of PSMA.