Fluoride contamination in groundwater has raised serious health concerns. The traditional technologies available for fluoride removal are having several shortcomings. Hence, biosorption is considered as a potential eco-friendly alternative to the existing methods. In the present work, several soil samples were collected from different fluoride contaminated regions of Pavagada taluk, Tumkur district, Karnataka, India. These samples were used as a source to isolate fungi with distinct morphological characteristics. The four fluoride-resistant fungal species were identified as Penicillium camemberti SIT-CH-1, Aspergillus ficuum SIT-CH-2, Aspergillus terreus SIT-CH-3, and Aspergillus flavipes SIT-CH-4 and were tested for their biosorption properties. The effect of parameters such as initial fluoride concentration, pH, contact time, and biosorbent dosage were optimized using a Central Composite design (CCD) of Response Surface Methodology (RSM). Penicillium camemberti SIT-CH-1 showed better fluoride removal than the other three fungal species. The pseudo-second order kinetic model provided the best fit for all the four fungal species for fluoride biosorption.