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Background Squash leaf curl virus (SLCV) is efficiently transmitted and spread by the whitefly, Bemisia tabaci (Gennadius), which is the only vector that transmits begomoviruses naturally causing huge crop losses through feeding damage. The widespread use of chemical insecticides to control the whitefly B. tabaci has become extremely hazardous to the environment. Alternative methods such as biological control have been advocated. Entomopathogenic fungi (EPF) have been found as promising whitefly bio-pesticides. Results Naturally infected squash plants that showed symptoms of squash leaf curl disease were collected from Giza Governorate, Egypt. SLCV was detected using a PCR assay using coat protein-specific primers and generated an amplicon of 419 bp. Multiple sequence alignment showed that the SLCV-Giza isolate has a significant identity of 99.2% with the SLCV-Mx:BCS: La Paz isolate from Mexico and 99% with the SLQV.Q2521 isolate from Egypt. Phylogenetic analysis showed that SLCV-Giza is closely related to the SLCV-Mx:BCS: La Paz isolate from Mexico. The whitefly transmission test revealed that the virus transmitted to an extent of 13.3% and reached 100% of transmission using 15–20 viruliferous whiteflies; while the efficiency of syringe injection was (60%). B. tabaci newly emerge adults were able to acquire and transmit SLCV after an Acquisition Access Period (AAP) of 15 and 30 min by low rates of 13.3 and 22.2%, respectively. The transmission rate was increased gradually to reach the maximum of 100% after 24, 48, and 72 h (AAP). B. tabaci was able to inoculate SLCV after an Inoculation Access Period (IAP) of 15 and 30 min with rates of 46.7 and 62.2%. The whitefly was allowed to acquire SLCV from a squash plant (virus source) treated previously with EPF (Beauveria bassiana) and allowed to transmit the virus to the test plants. The transmission effectiveness of viruliferous whitefly was lower (33.4%) than that of non-treated whitefly (100%). The transmission efficiency was decreased on the second day by 6.8% and by the third day by 2.2% of treatment with the EPF. The results were validated by PCR assay for SLCV from tested squash plants and the PCR did not reveal specific amplification. Conclusions The use of EPF (B. bassiana) for B. tabaci control had a direct impact on SLCV accumulation and transmission.