Heavy metal (HM) pollution is a worldwide environmental issue. Given the urgent need to develop more powerful approaches for effective phytoremediation of HMs, isolation of novel endophytic strains from hyperaccumulator plants having potent HM tolerance is the main objective in this research. Moreover, the recovered strains were characterized and subjected to radiation mutagenesis to enhance their tolerance to HMs. Among 105 isolates, Alternaria alternata AUMC14431 was identified as the most effective Cd+2 tolerant strain having high recorded tolerance index (TI) (76.24%); in addition, the recorded minimum inhibitory concentration (MIC) was 300ppm. Meanwhile, Chaetomium globosum AUMC14432 was identified as the most effective Pb+2 and Ni+2 tolerant strain having high recorded TI (97.46 and 93.34%, respectively); in addition, the evaluated MICs were 250 and 200ppm, respectively. UV and gamma irradiation of the tested strains enhanced their Cd+2 and Pb+2 tolerance significantly (P ≤ 0.05). Meanwhile, irradiation had a negative impact on Ni+2 tolerance of C. globosum. The mutation incidence at the molecular level arising from exposure to irradiation was investigated. Genomic DNA of both the wild and mutated endophytic strains were isolated followed by random amplified polymorphic DNA (RAPD-PCR) analysis, using two short primers. A remarkable difference in DNA gel pattern between the wild type and mutated strains was observed. In conclusion, the novel isolated and irradiated endophytic strains, A. alternata S5 and C. globosum El26, having high efficiency in Cd+2 and Pb+2 tolerance, respectively, are considered to be prospective and powerful bioremediation candidates for potential application in microbially assisted phytoremediation.