A study was conducted on populations of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), infected with the microsporidia Vairimorpha invictae Jouvenaz and Ellis (Microsporidia: Burenellidae) and Thelohania solenopsae Knell, Allen, and Hazard (Microsporidia: Thelohaniidae). Fire ant populations and microsporidia prevalence were monitored three to five times per year for 3–4 yr in eight field plots in northern Argentina. The mean population index per plot showed an overall reduction of 69%. The percentage of infection with V. invictae and T. solenopsae showed fluctuations that ranged from 29.2 to 1.4% and 13.6 to 2.6%, respectively. The highest infection rates were observed at the beginning of the study. A total of 394 colonies were sampled during the study: 325 (82.5%) were healthy and 69 (17.5%) were infected with microsporidia. The proportion of infected colonies with brood was 81% (56/69), similar to the proportion of healthy colonies with brood (78%; 255/325). The proportion of infected and healthy colonies in the population index categories was significantly different. Of the infected colonies with brood, 49.3% were medium and 1.4% were large in size. In contrast, healthy colonies were generally larger, with 29.7 and 10.4% being medium and large, respectively. The general environmental conditions in the area of the plots were appropriate for fire ant population growth; consequently, they do not explain the overall reduction in the populations. These results, combined with additional evidence reported previously, suggest that infection with V. invictae and T. solenopsae has a deleterious effect on native populations of S. invicta.