There is a need to develop low-cost methods for larviculture that emphasize disease prevention. We evaluated stagnant water larviculture in flounder Paralichthys olivaceus using rotifer amictic eggs treated with glutaraldehyde. Growth and survival of the larvae were similar to in larvae reared by standard running water larviculture with daily feeding of rotifers (control). In the experimental group, the n-3 highly unsaturated fatty acid content of rotifers in the rearing water was higher than in the enriched rotifers fed to the control group. We also observed a similar pattern for the fatty acid composition of flounder larvae. The viable bacterial counts associated with rotifers in stagnant water were lower than those associated with enriched rotifers, and there were fewer bacterial genera. However, the viable bacterial count was the same or higher in the experimental rearing water compared with that in the control group. The α-Proteobacteria and Cytophaga–Flavobacterium–Bacteroidetes group dominated the bacterial community structure after larval hatching. To our knowledge, this is the first time that stagnant water larviculture, using only disinfected amictic eggs, has been successfully demonstrated on a mass production scale. This method appears to offer benefits including improved nutrition, more favorable bacterial communities, and lower cost.