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(1) Background: Microinjection of zebrafish (<i>Danio rerio</i>) embryos offers a promising model for studying the virulence and potential environmental risks associated with <i>Pseudomonas aeruginosa</i>. (2) Methods: This work aimed to develop a <i>P. aeruginosa</i> infection model using two parallel exposition pathways on zebrafish larvae with microinjection into the yolk and the perivitelline space to simultaneously detect the invasive and cytotoxic features of the examined strains. The microinjection infection model was validated with 15 environmental and clinical strains of <i>P. aeruginosa</i> of various origins, antibiotic resistance profiles, genotypes and phenotypes: both exposition pathways were optimized with a series of bacterial dilutions, different drop sizes (injection volumes) and incubation periods. Besides mortality, sublethal symptoms of the treated embryos were detected and analyzed. (3) Results: According to the statistical evaluation of our results, the optimal parameters (dilution, drop size and incubation period) were determined. (4) Conclusions: The tested zebrafish embryo microinjection infection model is now ready for use to determine the in vivo virulence and ecological risk of environmental <i>P. aeruginosa</i>.