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In our work to enhance the efficiency of silicon nanoparticles (Si-NPs) production, we suggested using preliminary nano- or microstructured silicon instead of usually used bulk crystalline silicon targets: porous silicon films, silicon nanowires arrays and mechanically grinded silicon microparticles (1–8 μm in size) were used as targets at irradiation by picosecond laser pulses (1064 nm, 34 ps, 1–16 mJ). As a result, variation of the silicon-based targets morphology and buffer liquid (water or ethanol) allowed to fabricate Si-NPs with mean sizes from 25 to 200 nm and the high degree of crystallinity. Additionally, we revealed that at pulsed laser fragmentation of the silicon microparticles in water, the Si-NPs size distributions strongly depend on their initial concentration. An appropriate simulation of propagation of a focused laser beam in a scattering suspension of silicon microparticles was performed for their different mass concentrations and allowed to explain the obtained results. The crystallinity of the Si-NPs ensures fluorescence with a maximum in the red spectral range that indicates the prospects of the fabricated nanoparticles as fluorescence markers in optical bioimaging. Another way to vizualize structiral inhomogenities in biological tissuses is using effective Mie scattering in the red and near infrared spectral ranges when the fabricated suspensions may be applied as contrast agents in the optical coherence tomography technique. Experiments with suspensions drops administered on agar gel surfaces confirm such possibility. Using the extracted scattering and absorption parameters of the Si-NPs suspensions, the heating of tumor tissue (basal-cell carcinoma) with embedded nanoparticles was numerically modelled. It was demonstrated that irradiation by a laser beam with the wavelength 633 nm allows to obtain a temperature contrast between tumor and surrounding normal tissues about 5 K, which is suitable for photohyperthermia. Photoinduced heating of agar phantoms with embedded Si-NPs confirmed this tendency. Thus, the obtained results allow to conclude that the Si-NPs fabricated via laser ablation and fragmentation are promising both optical bioimaging modalities and in photohyperthermia of tumors.