In this study, eight fungal strains were isolated from the rhizosphere of Oenothera picensis Phil., a plant that grows naturally in soil contaminated with metal (loid)s in central Chile. The capacity of the strains to tolerate copper and zinc was evaluated, and their effect on the expression of genes related to metal tolerance in Solanum lycopersicum co-inoculated with arbuscular mycorrhizal fungi. Among the isolates, Lecythophora sp. and Hormonema viticola showed the highest tolerance to Cu (1150 mg kg|−1) and Zn (3200 mg kg−1), respectively. We detected the highest lactic and oxalic acid production lactic and oxalic acid production in Lecythophora sp. against Cu contamination. In contrast, a higher malic, lactic, and citric acid production was detected in H. viticola against Zn contamination. Regarding the expression involved in metal tolerance, we showed that both fungi reduce the expression of the genes MT, PCS, HSP, and NRAMP in S. lycopersicum growing under metal stress. We did not observe a significant positive effect of the co-inoculation with the arbuscular mycorrhizal fungus Rhizophagus irregularis in the relative expression genes in leaves, but a positive impact was detected in roots. The results showed that the co-inoculation of rhizosphere metal-tolerant fungi isolated from metallophyte species and mycorrhizal fungi decreased the expression of metal tolerance genes in S. lycopersicum growing under toxic Cu and Zn levels.