In an ant colony, a large number of nestmates with a similar gene pool coexist, making them more vulnerable to pathogenic attacks. These pathogens influence the behavior and physiology of the fire ant Solenopsis invicta Buren. Here, we evaluated the impact of entomopathogenic fungi (EPF) Metarhizium anisopliae on the behavior (locomotion and foraging) and physiology (biological molecules, anti-fungal activity, and survival) of S. invicta. Distance traveled and velocity significantly decreased, while turn angle and angular velocity significantly increased in ants exposed to a higher concentration of M. anisopliae compared to ants exposed to control after 36 h, which showed disturbed locomotion. Fungus infection significantly affected the foraging behavior of ants. Fungus-exposed ants spent significantly less time in the food zone (area with food) than in the inner zone (area without food). The activities of 4 enzymes, peroxidase, glutathione-S-transferase, hydrogen peroxide (H2O2), and carboxylesterase were significantly decreased. In contrast, catalase and anti-fungal activities were increased after fungal exposure compared to the control. The activity of acetylcholinesterase, which hydrolyses the important neurotransmitter acetylcholine, also decreased after fungal application compared to the control. Survival of ants was also significantly reduced after fungus infection compared to the control. Our findings help to understand the influence of M. anisopliae on the behavior and physiology of S. invicta, which will help in the management of S. invicta using the EPF M. anisopliae.