One of the most important aspects of shaping the antibacterial properties of nano-mixed cementitious composites is the proper dispersion of nanoparticles (NPs) to ensure their sustainable application. Therefore, in the presented article special attention was paid to the method of mixing zinc oxide NPs with the cement matrix. A two-stage modification of the cement matrix was used; in the first stage, zinc oxide NPs were introduced directly in dry form into the cement binder or mixed mechanically, with ultrasound and combined mechanically and with ultrasound. Then, in the second stage, a superplasticizer was added to the mixing methods mentioned above. It was shown that the combined use of premixing and then mixing with superplasticizer influences better dispersion of zinc oxide in the cement matrix, which in turn translates into higher mechanical parameters, better microstructure compaction and resistance to microorganisms. The most homogeneous mixture was obtained with combined mechanical and ultrasonic mixing. On the other hand, better antimicrobial properties were exhibited by composites obtained only by mechanical mixing.