We surveyed 154 sites in north-central Argentina and sampled 2,528 fire ant colonies for the presence and intracolonial prevalence of the microsporidium, Vairimorpha invictae Jouvenaz & Ellis, in the red imported fire ant, Solenopsis invicta Buren. The concentrations of meiospores and binucleate spores were quantified in workers and sexuals; and the occurrence and intracolonial prevalence of dual infections with Thelohania solenopsae Knell, Allen & Hazard were studied. To study the effect of V. invictae in infected colonies of S. invicta, we compared the proportion of infected living workers to the proportion of infected dead workers, and compared the survival of uninfected and infected workers. V. invictae occurred at 13% of the sites and 2.3% of the colonies. At times, the disease reached epizootic levels in certain areas. We found vegetative stages in 4.8–52.3% of eggs, larvae, pupae, and queens, meiospores in 4–56.3% of pupae and mature stages, and binucleate spores in 9.5–63% of all life stages, except eggs. Evidence for transovarial transmission is provided. The percentage of sexual males infected was significantly higher than that of sexual females (44.9 versus 15.9%, respectively). Dual infections (V. invictae T. solenopsae) occurred in 0.24% of the colonies. V. invictae was present in 9.3% of living workers and in 56.7% of dead workers. Mortality rates of workers from Vairimorpha-infected colonies were higher than those of workers from uninfected colonies. Survival times of infected workers were 18.8–31.7% less than those of uninfected workers. The studies reported here contribute to the evaluation of V. invictae for use as a classical biological control agent against the red imported fire ant in the United States.