Bacterial biosynthesis of quantum dots (QDs) offers a green alternative for the production of nanomaterials with superior properties, such as tunable size dependent emission spectra and a long fluorescence lifetime. In this study, we have achieved intracellular production of PbS QDs using Pseudomonas aeruginosa ATCC 27853. The characterization of these PbS QDS was performed by different techniques, such as Ultraviolet–visible (UV–Vis) spectroscopy, photoluminescence (PL), X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDX) and particle size analysis (DLS). The obtained results confirmed the synthesis of PbS QDs. The PbS QDs showed absorption peaks at 1088 nm (ultraviolet–visible spectrometry) and a luminescence peak at 1572 nm. In addition, the intracellular biosynthesized PbS QDs showed a particle size in the range 3.47–11.45 nm, which is consistent with DLS and sphere-shaped nanocrystals with good crystallinity and a cubic cristalline structure including pure Pb and S elements. Biosynthesized PbS QDs showed antibacterial activity against Proteus mirabilis ATCC 25933 and Escherichia coli ATCC 25922 from Gram (−) bacteria and Bacillus cereus NRLL B-3008 and Micrococcus luteus ATCC 10240 from Gram (+) bacteria. Besides, these current results obtained from the cleavage studies revealed that PbS QDs do not show DNA cleavage activity. Consequently, the microorganism-based intracellular method allows an economic and environmentally friendly way to obtain PbS QDs with superior optical properties and they have a potential to be used in healthcare applications.