Several different fungi can cause tar leaf spot diseases in maple trees, including three fungi of the genus Rhytisma. Rhytisma acerinum (Pers.) Fries is an ascomycete that forms black stromata known as tar spot on the adaxial surface of the leaves of Acer species. The tar spot (R. acerinum) disease has been increasing in incidence and severity in maples of Hyrcanian forests, northern Iran, in recent years. One of the best ways to manage infestations by R. acerinum is through adequate biotechnical techniques. The isolation of fungal spore colonies was evaluated using different dosages of Oxywet 10% (50, 100, 200, 500 µl), Gentamicin 5% (100, 200, 400; 1,000 µl), and Amoxicillin antibiotics 20% (25, 50, 100, 250 µl) in 100 ml of distilled water in each treatment. All possible combinations of single doses were applied using light and dark treatments. In light conditions, it appears that the Oxywet (200 µl) had the significant effect on controlling R. acerinum. Reduced fungal growth, coefficient and inhibition of fungal growth were observed in the light treatment. The other antibiotics (Gentamicin, Amoxicillin) were not so effective in controlling this pathogen. Results of spore germination showed a significant difference between all treatments. All treatments were tested in pure cultures in the laboratory only. The results obtained cannot be expected of the same effectiveness in open field trials.