Nanoprecipitation is a facile and efficient approach to the assembly of loaded polymer nanoparticles (NPs) for applications in bioimaging and targeted drug delivery. Their successful use in clinics requires reproducible and scalable synthesis, for which microfluidics appears as an attractive technique. However, in the case of nanoprecipitation, particle formation depends strongly on mixing. Here, we compare 5 different types of microfluidic mixers with respect to the formation and properties of poly(D-L-lactide-co-glycolide) (PLGA) and poly(methyl methacrylate) NPs loaded with a fluorescent dye salt: a cross-shaped mixer, a multilamination mixer, a split and recombine mixer, two herringbone mixers, and two impact jet mixers. Size and fluorescence properties of the NPs obtained with these mixers are evaluated. All mixers, except the cross-shaped one, yield NPs at least as small and fluorescent as those obtained manually. Notably in the case of impact jet mixers operated at high flow speeds, the size of the NPs could be strongly reduced from >50 nm down to <20 nm. Surprisingly, the fluorescence quantum yield of NPs obtained with these mixers also depends strongly on the flow speed, increasing, in the case of PLGA, from 30 to >70%. These results show the importance of precisely controlling the assembly conditions for loaded polymer NPs. The present work further provides guidance for choosing the optimal microfluidic setup for production of nanomaterials for biomedical applications.