Carbofuran is a pesticide used against insects and nematodes. It is a common environmental pollutant associated with agricultural activities. Carbofuran has harmful effects on target and non-target organisms through the accumulation of acetylcholine at the junction of the nerve cell and the receptor sites system by inhibiting acetylcholine esterase. Since the Kingdom of Saudi Arabia is one of the countries that use the pesticide carbofuran in agricultural applications, it inevitably suffers from this worsening environmental problem. Therefore, removal of this compound from the environment is a necessity to ensure human health. In this study, 20 fungal isolates were isolated from pesticide-contaminated agricultural soils. Three fungal isolates (KKU-F5, KKU-F11, KKU-F17) were selected that have the ability to tolerate and utilise carbofuran. The exact identification of these isolates was determined using ITS1-5.8S rRNA-ITS2 region amplification and sequence determination. Alignment results and the comparison of ITS1-5.8S rRNA-ITS2 region sequences of the isolates to ITS1-5.8S rRNA-ITS2 sequences available in the GenBank database, as well as the phylogenetic analysis, confirmed the accurate identification of the isolates KKU-F5, KKU-F11 and KKU-F17 as Fusarium polyphialidicum, Fusarium oxysporum and Fusarium verticillioides, respectively. The three selected fungi demonstrated a high growth rate on 3 gm/L carbofuran, which is twice the recommended dose of carbofuran, after an incubation period of seven days. The percentage of carbofuran removal determined by HPLC analysis for the isolates KKU-F 5, KKU-F11 and KKU-F17 was 88%, 77% and 90.10% respectively, within this period. RAPD-PCR fingerprinting was performed to determine the differentiation at the molecular level within the three Genetics and Molecular Research 17 (1): gmr16039900 carbofuran-degrading Fusarium isolates (KKU-F5, KKU-F11 and KKU-F17). The RAPD-PCR results exhibited different RAPD banding profiles indicating that the three Fusarium isolates were different from each other and could be clearly distinguished by their amplification patterns.