Three studies were conducted to assess the effects of the entomopathogen Thelohania solenopsae on polygynous, red imported fire ant, Solenopsis invicta Buren, colonies. A total of 57 of 122 queens (46.7%) from nine, field-collected, polygyne, S. invicta colonies, was infected with T. solenopsae. Infection rate of queens for each colony ranged from 25 to 75%. Laboratory colonies of polygyne S. invicta, with three to 12 queens, were inoculated and infected with T. solenopsae. Brood levels in all infected colonies declined to 0 after 26–52 wk. Brood did not reappear in any of the colonies after 3–11 wk, even though in two of the eight infected colonies, five fertile queens that were uninfected were recovered. Thus, polygyne, S. invicta colonies infected with T. solenopsae, which were confined and isolated under laboratory conditions, did not recover. Field plots that contained polygynous S. invicta colonies, which were infected with T. solenopsae, were monitored over a 2-yr period. Infection rates increased during the study and reached a maximum of 93%. Fire ant nest density and colony sizes fluctuated over time, with maximum reduction of 63% per plot. In general, fire ant reductions were attributed to smaller colony sizes. T. solenopsae infections in polygynous S. invicta can result in a slow colony decline and death. Under field conditions, the prolonged colony death may mask the impact of T. solenopsae by allowing for concurrent reinfestations.