Musca domestica is a major problem in poultry production where management has relied mostly on pesticides, which eventually have led to the development of widespread resistance in insect populations and environmental contamination. In this study, we explored the use of entomopathogenic fungi for the control of houseflies in Brazil. A total of 10 isolates of different fungal pathogens were tested on the larval stages of M. domestica, which were originally derived from a commercial aviary. Among the test fungi, Metarhizium anisopliae (sensu stricto) CG46 and CG30 were the most virulent isolates, causing up to 60% mortality at day 10, but only CG46 was selected for a field trial as it attained higher sporulation on larval cadavers. A commercial product based on the M. anisopliae isolate ESALQ-1037 (Metarril®) was also field-tested using standard treatment procedures. After spraying both fungi in the aviary manure, there was a significant reduction (twofold lower) in adult density of M. domestica captured per trap compared to the control shed. Therefore, our results indicate that M. anisopliae is a promising biological control agent for M. domestica larvae, but new strategies for fungal application and appropriate formulation need to be developed to improve its effectiveness in poultry environments.